Butyrylcholinesterase variant polypeptides with increased catalytic efficiency and methods of use

ABSTRACT

The invention provides a butyrylcholinesterase variant having increased cocaine hydrolysis activity as well as the corresponding encoding nucleic acid. The invention further provides methods of hydrolyzing a cocaine-based butyrylcholinesterase substrate as well as methods of treating a cocaine-induced condition with the invention variant.

This application is a continuation-in-part of U.S. Ser. No. 10/324,466,filed Dec. 20, 2002, which claims benefit of provisional applicationSer. No. 60/560,741, filed Dec. 20, 2001, which was converted to aprovisional application by Petition Under 37 C.F.R. §1.53(c)(2)(i), andis incorporated herein by reference in its entirety.

This invention was made with government support under grant number 1R01DA011707 awarded by the National Institutes of Health. The United StatesGovernment has certain rights in this invention.

BACKGROUND OF THE INVENTION

The present invention relates generally to the fields of computationalchemistry and molecular modeling and, more specifically, tobutyrylcholinesterase polypeptide variants with increased catalyticefficiency.

Cocaine abuse is a significant social and medical problem in the UnitedStates as evidenced by the estimated 3.6 million chronic users. Cocaineabuse often leads to long-term dependency as well as life-threateningoverdoses. However, no effective antagonist is currently available thatcombats the reinforcing and toxic effects of cocaine.

One difficulty in identifying an antagonist to treat cocaine abusearises largely from the narcotic's mechanism of action. Specifically,cocaine inhibits the, re-uptake of neurotransmitters resulting inover-stimulation of the reward pathway. It is this over-stimulation thatis proposed to be the basis of cocaine's reinforcing effect. Inaddition, at higher concentrations, cocaine interacts with multiplereceptors in both the central nervous and cardiovascular systems,leading to toxicities associated with overdose. Because of thismultifarious mechanism of action of cocaine, it is difficult to identifyselective antagonists to treat both the reinforcing and toxic effects ofcocaine. Additionally, antagonists that block cocaine's binding to itsreceptors tend to display many of the same deleterious effects ascocaine.

Recently, alternative treatment strategies based on intercepting andneutralizing cocaine in the bloodstream have been proposed. For example,dopamine D1, D2, and D3 antagonists affect the reinforcing potency ofcocaine in the rat model, these antagonists display a narrow range ofeffective doses and the extent of decrease in cocaine potency is quitesmall. In addition, these dopamine antagonists produce profounddecreases in other behaviors when the doses are increased only slightlyabove the levels that display an effect on cocaine self-administrationbehavior.

A separate treatment strategy involves partial protection against theeffects of cocaine using antibody-based approaches. Limitations ofimmunization approaches include the stoichiometric depletion of theantibody following the binding of cocaine. The use of a catalyticantibody, which metabolizes cocaine in the bloodstream, partiallymitigates this problem by degrading and releasing cocaine, permittingbinding of additional cocaine. However, the best catalytic antibodyidentified to date metabolizes cocaine significantly slower thanendogenous human serum esterases.

In vivo, cocaine is metabolized by three principal routes: 1)N-demethylation in the liver to form norcocaine, 2) hydrolysis by serumand liver esterases to form ecgonine methyl ester, and 3) nonenzymatichydrolysis to form benzoylecgonine. In humans, norcocaine is a minormetabolite, while benzoylecgonine and ecgonine methyl ester account forabout 90% of a given dose. The metabolites of cocaine are rapidlycleared and appear not to display the toxic or reinforcing effects ofcocaine. Low serum levels of butyrylcholinesterase have been correlatedwith adverse physiological events following cocaine overdose, providingfurther evidence that butyrylcholinesterase accounts for the cocainehydrolysis activity observed in plasma. Human plasma obtained fromindividuals with a defective version of the butyrylcholinesterase genehas been shown to have little or no ability to hydrolyze cocaine invitro, and the hydrolysis of cocaine in plasma of individuals carryingone defective and one wild type copy of the butyrylcholinesterase genehas been shown to proceed at one-half the normal rate. Therefore, it hasbeen suggested that individuals with defective versions of thebutyrylcholinesterase gene are at higher risk for life-threateningreactions to cocaine. Recently, administration of butyrylcholinesterasehas been demonstrated to confer limited protection against cocaineoverdose in mice and rats.

Although administration of butyrylcholinesterase provides some effectagainst cocaine toxicity in vivo, it is not an efficient catalyst ofcocaine hydrolysis. The low cocaine hydrolysis activity of wild-typebutyrylcholinesterase requires the use of prohibitively large quantitiesof purified enzyme for therapy.

A number of naturally occurring human butyrylcholinesterases as well asspecies variations are known, none of which exhibits increased cocainehydrolysis activity. Similarly, although a variety of recombinantlyprepared butyrylcholinesterase mutations have been tested for increasedcocaine hydrolysis activity, only one such mutant, termed A328Y, hasbeen identified that exhibits increased cocaine hydrolysis activity.Further butyrylcholinesterase mutations that lead to increased cocainehydrolysis activity need to be identified to permit clinical evaluationof butyrylcholinesterase.

Thus, there exists a need for recombinant butyrylcholinesterasepolypeptides capable of hydrolyzing cocaine significantly moreefficiently than wild-type butyrylcholinesterase. The present inventionsatisfies this need and provides related advantages as well.

SUMMARY OF THE INVENTION

The invention provides a butyrylcholinesterase variant polypeptidehaving increased cocaine hydrolysis activity as well as thecorresponding encoding nucleic acid. The invention further providesmethods of hydrolyzing a cocaine-based butyrylcholinesterase substrateas well as methods of treating a cocaine-induced condition with theinvention variant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows alignment of amino acid and nucleic acid sequences for allbutyrylcholinesterase variant alterations in their respective regions ofhuman butyrylcholinesterase.

FIG. 2 shows the amino acid sequence of human butyrylcholinesterase (SEQID NO: 44).

FIG. 3 shows the nucleic acid sequence of human butyrylcholinesterase(SEQ ID NO: 43).

FIG. 4 shows an amino acid sequence alignment of human wild-type (SEQ IDNO: 44), human A variant (SEQ ID NO: 45), human J variant (SEQ ID NO:46), human K variant (SEQ ID NO: 47), horse (SEQ ID NO: 48), cat (SEQ IDNO: 49) and rat butyrylcholinesterase variants (SEQ ID NO: 50).

FIG. 5 shows (A) the correlation between the HPLC assay and the isotopetracer assay as demonstrated by plotting the quantitation of benzoicacid formation by both methods, and (B) the K_(m) for cocaine hydrolysisactivity of horse butyrylcholinesterase using the Lineweaver-Burkdouble-reciprocal plot.

FIG. 6 shows solid phase immobilization of wild-type (filled circles)and truncated (open circles) butyrylcholinesterase for measuring cocainehydrolysis activity.

FIG. 7 shows the use of multiple synthesis columns and codon-basedmutagenesis for the synthesis of focused libraries.

FIG. 8 shows the effect of pre-treatment with AME-359 (solid circles) orwild-type BChE (open circles) on cocaine-induced toxicity. AME-359exhibited statistically significant protection against cocaine(Chi-squared test; p<0.001).

FIG. 9 shows the effect of therapeutic treatment with AME-359 oncocaine-induced toxicity. AME-359 maintained full protection whenadministered at 8 minutes into the cocaine infusion (in particular,measured from the first set of slight convulsions) and decreased inability to protect when administered at later time points.

FIG. 10 shows plasma levels of wt BChE and AME-359 following an i.v.bolus of 1 mg/kg. Wild-type BChE pool I or pool II (open squares andopen circles, respectively) and AME-359 pool I or pool II (solid squaresand solid circles, respectively). BChE activity was determined byenzymatic assay utilizing butyrylthiocholine as the substrate.

FIG. 11 shows plasma levels of an intravenous bolus of cocaine aftertreatment with AME-359. Cocaine was administered at 10 mg/kg (opencircles) and AME-359 administered immediately at 0.01 mg/kg and 0.05mg/kg (solid circles and solid squares, respectively).

FIG. 12 shows the prophylactic effect of the butyrylcholinesterasevariant designated A328W/Y332M/S287G/F227A on cocaine-inducedconvulsions. The variant was administered at the indicated doses, 1minute prior to infusion of 30 mg/kg cocaine (2 mg/kg/min for 15minutes). The data is presented as mean±sem.*p<0.001 vs. control, 0.1mg/kg or 0.2 mg/kg variant-treated animals; ANOVA followed by Bonferronipost-test.

DETAILED DESCRIPTION OF THE INVENTION

This invention is directed to butyrylcholinesterase variant polypeptideshaving increased cocaine hydrolysis activity compared to naturallyoccurring human butyrylcholinesterase, as well as to their encodingnucleic acids. The invention also is directed to methods of hydrolyzinga cocaine-based butyrylcholinesterase substrate and to methods oftreating a cocaine-induced condition.

Cholinesterases are ubiquitous, polymorphic carboxylase Type B enzymescapable of hydrolyzing the neurotransmitter acetylcholine and numerousester-containing compounds. Two major cholinesterases areacetylcholinesterase and butyrylcholinesterase. Butyrylcholinesterasecatalyzes the hydrolysis of a number of choline esters as shown:

Butyrylcholinesterase preferentially uses butyrylcholine andbenzoylcholine as substrates. Butyrylcholinesterase is found inmammalian blood plasma, liver, pancreas, intestinal mucosa and the whitematter of the central nervous system. The human gene encodingbutyrylcholinesterase is located on chromosome 3 and over thirtynaturally occurring genetic variations of butyrylcholinesterase areknown. The butyrylcholinesterase polypeptide is 574 amino acids inlength and encoded by 1,722 base pairs of coding sequence. Threenaturally occurring butyrylcholinesterase variations are the typicalalleles referred to as A variant (SEQ ID NO: 45), the J variant (SEQ IDNO: 46) and the K variant (SEQ ID NO: 47), which are aligned in FIG. 4.The A variant has a D70G mutation and is rare (0.5% allelic frequency),while the J variant has an E497V mutation and has only been found in onefamily. The K variant has a point mutation at nucleotide 1615, whichresults in an A539T mutation and has an allelic frequency of around 12%in Caucasians.

In addition to the naturally-occurring human variations ofbutyrylcholinesterase, a number of species variations are known. Theamino acid sequence of cat butyrylcholinesterase is 88% identical withhuman butyrylcholinesterase (see FIG. 4). Of the seventy amino acidsthat differ, three are located in the active site gorge and are termedA277L, P285L and F398I. Similarly, horse butyrylcholinesterase has threeamino acid differences in the active site compared with humanbutyrylcholinesterase, which are A277V, P285L and F398I (see FIG. 4).The amino acid sequence of rat butyrylcholinesterase contains 6 aminoacid differences in the active site gorge, which are A277K, V280L,T284S, P285I, L286R and V288I (see FIG. 4).

Naturally occurring human butyrylcholinesterase variations, speciesvariations as well as recombinantly prepared mutations have previouslybeen described by Xie et al., Molecular Pharmacology 55:83-91 (1999).Recombinant human butyrylcholinesterase mutants that have been testedfor increased cocaine hydrolysis activity include mutants with thefollowing single or multiple changes: N68Y/Q119/A277W, Q119/V288F/A328Y,Q119Y, E197Q, V288F, A328F, A328Y, F329A and F329S. Out of thesemutants, the only butyrylcholinesterase mutant identified that exhibitsincreased cocaine hydrolysis activity is the A328Y mutant, which has aTyrosine (Y) rather than an Alanine (A) at amino acid position 328 andexhibits a four-fold increase in cocaine hydrolysis activity compared tohuman butyrylcholinesterase (Xie et al., supra, 1999).

The invention provides butyrylcholinesterase variant polypeptidesencompassing the same or substantially the same amino acid sequence asshown as SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42 and 52 and functional fragments ofbutyrylcholinesterase variant polypeptides encompassing the same orsubstantially the same amino acid sequence as shown as SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42and 52.

The butyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence as shown as SEQ ID NO: 2, orfunctional fragment thereof, has a twenty-four-fold increase in cocainehydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 4, orfunctional fragment thereof, has a ten-fold increase in cocainehydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 6, orfunctional fragment thereof, has a sixteen-fold increase in cocainehydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 8, orfunctional fragment thereof, has a eight-fold increase in cocainehydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 10, orfunctional fragment thereof, has a one-hundred-fold increase in cocainehydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 12, orfunctional fragment thereof, has a one-hundred-fold in cocainehydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 14, orfunctional fragment thereof, has a ninety-seven-fold in cocainehydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 16, orfunctional fragment thereof, has a ninety-one-fold in cocaine hydrolysisactivity relative to butyrylcholinesterase. The butyrylcholinesterasevariant polypeptide encompassing the same or substantially the sameamino acid sequence shown as SEQ ID NO: 18, or functional fragmentthereof, has a sixty-eight-fold in cocaine hydrolysis activity relativeto butyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 20, or functional fragment thereof, has an increasedcocaine hydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 22, orfunctional fragment thereof, has an increased cocaine hydrolysisactivity relative to butyrylcholinesterase. The butyrylcholinesterasevariant polypeptide encompassing the same or substantially the sameamino acid sequence shown as SEQ ID NO: 24, or functional fragmentthereof, has an increased cocaine hydrolysis activity relative tobutyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 26, or functional fragment thereof, has an increasedcocaine hydrolysis activity relative to butyrylcholinesterase. Thebutyrylcholinesterase variant polypeptide encompassing the same orsubstantially the same amino acid sequence shown as SEQ ID NO: 28, orfunctional fragment thereof, has a four-fold in cocaine hydrolysisactivity relative to butyrylcholinesterase. The butyrylcholinesterasevariant polypeptide encompassing the same or substantially the sameamino acid sequence shown as SEQ ID NO: 30, or functional fragmentthereof, has a four-fold increase in cocaine hydrolysis activityrelative to butyrylcholinesterase. The butyrylcholinesterase variantpolypeptide encompassing the same or substantially the same amino acidsequence shown as SEQ ID NO: 32, or functional fragment thereof, has atwo-fold increase in cocaine hydrolysis activity relative tobutyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 34, or functional fragment thereof, has a three-foldincrease in cocaine hydrolysis activity relative tobutyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 36, or functional fragment thereof, has a two-foldincrease in cocaine hydrolysis activity relative tobutyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 38, or functional fragment thereof, has a two-foldincrease in cocaine hydrolysis activity relative tobutyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 40, or functional fragment thereof, has aone-and-a-half-fold increase in cocaine hydrolysis activity relative tobutyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 42, or functional fragment thereof, has atwo-and-a-half-fold increase in cocaine hydrolysis activity relative tobutyrylcholinesterase. The butyrylcholinesterase variant polypeptideencompassing the same or substantially the same amino the amino acidsequence shown as SEQ ID NO: 52, or functional fragment thereof, has aone-hundred-fold increase in cocaine hydrolysis activity relative tobutyrylcholinesterase.

The butyrylcholinesterase variant polypeptides of the invention holdsignificant clinical value because of their capability to hydrolyzecocaine at a higher rate than any of the known naturally occurringvariants. It is this increase in cocaine hydrolysis activity thatenables a much more rapid response to the life-threatening symptoms ofcocaine toxicity that confers upon the butyrylcholinesterase variantpolypeptides of the invention their therapeutic value. Thebutyrylcholinesterase variant polypeptides of the invention have two ormore amino acid alterations in regions determined to be important forcocaine hydrolysis activity.

As used herein, the term “butyrylcholinesterase” is intended to refer toa polypeptide having the sequence of naturally occurring humanbutyrylcholinesterase shown as SEQ ID NO: 44.

As used herein, the term “butyrylcholinesterase variant” is intended torefer to a molecule that is structurally similar to abutyrylcholinesterase, but differs by at least one amino acid from thebutyrylcholinesterase shown as SEQ ID NO: 44. A butyrylcholinesterasevariant is structurally similar to the butyrylcholinesterase shown asSEQ ID NO: 44, but exhibits increased cocaine hydrolysis activity. Forexample, the cocaine hydrolysis activity of a butyrylcholinesterasevariant polypeptide of the invention can be increased by a factor of 5,6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 24, 28, 32, 36, 40, 80, 100 or more.

A butyrylcholinesterase variant polypeptide can have a one, two, three,four, five, six or more amino acid alterations compared tobutyrylcholinesterase. A specific example of a butyrylcholinesterasevariant polypeptide has the amino acids Tryptophan and Methionine atpositions 328 and 332, respectively, of which the amino acid sequenceand encoding nucleic acid sequence is designated as SEQ ID NOS: 2 and 1,respectively. Additional examples of butyrylcholinesterase variantpolypeptides are the butyrylcholinesterase variant polypeptide havingthe amino acids Tryptophan and Proline at positions 328 and 332,respectively of which the amino acid sequence and nucleic acid sequenceare described herein and designated SEQ ID NOS: 4 and 3, respectively;the butyrylcholinesterase variant polypeptide having the amino acidsTryptophan and Leucine at positions 328 and 331, respectively, of whichthe amino acid sequence and nucleic acid sequence are described hereinand designated SEQ ID NOS: 6 and 5, respectively; thebutyrylcholinesterase variant polypeptide having the amino acidsTryptophan and Serine at positions 328 and 332, respectively, of whichthe amino acid sequence and nucleic acid sequence are described hereinand designated SEQ ID NOS: 8 and 7, respectively; thebutyrylcholinesterase variant polypeptide having the amino acids Serine,Alanine, Glycine, Tryptophan and Methionine at positions 199, 227, 287,328 and 332, respectively, of which the amino acid sequence and nucleicacid sequence are described herein and designated SEQ ID NOS: 10 and 9,respectively; the butyrylcholinesterase variant polypeptide having theamino acids Serine, Alanine, Glycine and Tryptophan at positions 199,227, 287 and 328, respectively, of which the amino acid sequence andnucleic acid sequence are described herein and designated SEQ ID NOS: 12and 11, respectively; the butyrylcholinesterase variant polypeptidehaving the amino acids Serine, Glycine and Tryptophan at positions 199,287 and 328, respectively, of which the amino acid sequence and nucleicacid sequence are described herein and designated SEQ ID NOS: 14 and 13,respectively; the butyrylcholinesterase variant polypeptide having theamino acids Alanine, Glycine and Tryptophan at positions 227, 287 and328, respectively, of which the amino acid sequence and nucleic acidsequence are described herein and designated SEQ ID NOS: 16 and 15,respectively; the butyrylcholinesterase variant polypeptide having theamino acids Alanine and Tryptophan at positions 227 and 328,respectively, of which the amino acid sequence and nucleic acid sequenceare described herein and designated SEQ ID NOS: 17 and 18, respectively;the butyrylcholinesterase variant polypeptide having the amino acidSerine at position 332, of which the amino acid sequence and nucleicacid sequence are described herein and designated SEQ ID NOS: 20 and 19,respectively; the butyrylcholinesterase variant polypeptide having theamino acid Methionine at position 332, of which the amino acid sequenceand nucleic acid sequence are described herein and designated SEQ IDNOS: 22 and 21, respectively; the butyrylcholinesterase variantpolypeptide having the amino acid Proline at position 332, of which theamino acid sequence and nucleic acid sequence are described herein anddesignated SEQ ID NOS: 24 and 23, respectively; thebutyrylcholinesterase variant polypeptide having the amino acid Leucineat position 331, of which the amino acid sequence and nucleic acidsequence are described herein and designated SEQ ID NOS: 26 and 25,respectively; the butyrylcholinesterase variant polypeptide having theamino acid Alanine at position 227, of which the amino acid sequence andnucleic acid sequence are described herein and designated SEQ ID NOS: 28and 27, respectively; the butyrylcholinesterase variant polypeptidehaving the amino acid Glycine at position 227, of which the amino acidsequence and nucleic acid sequence are described herein and designatedSEQ ID NOS: 30 and 29, respectively; the butyrylcholinesterase variantpolypeptide having the amino acid Serine at position 227, of which theamino acid sequence and nucleic acid sequence are described herein anddesignated SEQ ID NOS: 32 and 31, respectively; thebutyrylcholinesterase variant polypeptide having the amino acid Prolineat position 227, of which the amino acid sequence and nucleic acidsequence are described herein and designated SEQ ID NOS: 34 and 33,respectively; the butyrylcholinesterase variant polypeptide having theamino acid Tyrosine at position 227, of which the amino acid sequenceand nucleic acid sequence are described herein and designated SEQ IDNOS: 36 and 35, respectively; the butyrylcholinesterase variantpolypeptide having the amino acid Cysteine at position 227, of which theamino acid sequence and nucleic acid sequence are described herein anddesignated SEQ ID NOS: 38 and 37, respectively; thebutyrylcholinesterase variant polypeptide having the amino acidMethionine at position 227, of which the amino acid sequence and nucleicacid sequence are described herein and designated SEQ ID NOS: 40 and 39,respectively; the butyrylcholinesterase variant polypeptide having theamino acid Serine at position 199, of which the amino acid sequence andnucleic acid sequence are described herein and designated SEQ ID NOS: 42and 41, respectively; and the butyrylcholinesterase variant polypeptidehaving the amino acids Alanine, Glycine, Tryptophan and Methionine atpositions 227, 287, 328 and 332, respectively, of which the amino acidsequence and nucleic acid sequence are described herein and designatedSEQ ID NOS: 52 and 51, respectively.

As used herein, the term “polypeptide” is intended to mean two or moreamino acids covalently bonded together. A polypeptide of the inventionincludes small polypeptides having a few or several amino acids as wellas large polypeptides having several hundred or more amino acids.Usually, the covalent bond between the two or more amino acid residuesis an amide bond. However, the amino acids can be joined together byvarious other means known to those skilled in the peptide and chemicalarts. Therefore, a polypeptide, in whole or in part, can includemolecules which contain non-amide linkages between amino acids, aminoacid analogs, and mimetics. Similarly, the term also includes cyclicpeptides and other conformationally constrained structures. Apolypeptide also can be modified by naturally occurring modificationssuch as post-translational modifications, including phosphorylation,lipidation, prenylation, sulfation, hydroxylation, acetylation, additionof carbohydrate, addition of prosthetic groups or cofactors, formationof disulfide bonds, proteolysis, assembly into macromolecular complexes,and the like.

As described below, polypeptides of the invention also encompass, forexample, modified forms of naturally occurring amino acids such asD-stereoisomers, non-naturally occurring amino acids, amino acidanalogues and mimetics so long as such variants have substantially thesame amino acid sequence as the reference butyrylcholinesterase variantpolypeptide and exhibit about the same cocaine hydrolysis activity. Abutyrylcholinesterase variant polypeptide of the invention can have twoor more amino acid alterations. Furthermore, a butyrylcholinesterasevariant polypeptide of the invention can have one or more additionalmodifications that do not significantly change its cocaine hydrolysisactivity, but confer a desirable property such as increasedbiostability.

It is understood that the amino acid sequences of the invention can havea similar, non-identical sequence, and retaining comparable functionaland biological activity of the polypeptide defined by the referenceamino acid sequence. A variant polypeptide of the invention encompassessubstantially similar amino acid sequences that can have at least about75%, 80%, 82%, 84%, 86% or 88%, or at least 90%, 91%, 92%, 93% or 94%amino acid identity with respect to the reference amino acid sequence;as well as greater than 95%, 96%, 97%, 98% or 99% amino acid identity aslong as such polypeptides retain a biological activity of the referencebutyrylcholinesterase variant polypeptide. It is recognized, however,that polypeptides, or encoding nucleic acids, containing less than thedescribed levels of sequence identity arising as splice variants or thatare modified by conservative amino acid substitutions, or bysubstitution of degenerate codons also are encompassed within the scopeof the present invention.

A biological activity of a butyrylcholinesterase variant of theinvention is cocaine hydrolysis activity as described herein. Forexample, the butyrylcholinesterase variant A328W/Y332M designated SEQ IDNO: 2 exhibits about a twenty-four-fold increased cocaine hydrolysisactivity compared to butyrylcholinesterase; the butyrylcholinesterasevariant A328W/Y332P designated SEQ ID NO: 4 exhibits about a ten-foldincreased cocaine hydrolysis activity compared to butyrylcholinesterase;the butyrylcholinesterase variant A328W/V331L designated SEQ ID NO: 6exhibits about a sixteen-fold increased cocaine hydrolysis activitycompared to butyrylcholinesterase; the butyrylcholinesterase variantA328W/Y332S designated SEQ ID NO: 8 exhibits about a seven-foldincreased cocaine hydrolysis activity compared to butyrylcholinesterase;the butyrylcholinesterase variant A328W/Y332M/S287G/F227A/A199Sdesignated SEQ ID NO: 10 exhibits about a one-hundred-fold increasedcocaine hydrolysis activity compared to butyrylcholinesterase; thebutyrylcholinesterase variant A328W/S287G/F227A/A199S designated SEQ IDNO: 12 exhibits about a one-hundred-fold increased cocaine hydrolysisactivity compared to butyrylcholinesterase; the butyrylcholinesterasevariant A328W/S287G/A199S designated SEQ ID NO: 14 exhibits about aninety-seven-fold increased cocaine hydrolysis activity compared tobutyrylcholinesterase; the butyrylcholinesterase variantA328W/S287G/F227A designated SEQ ID NO: 16 exhibits about aninety-one-fold increased cocaine hydrolysis activity compared tobutyrylcholinesterase; the butyrylcholinesterase variant A328W/F227Adesignated SEQ ID NO: 18 exhibits about a sixty-eight-fold increasedcocaine hydrolysis activity compared to butyrylcholinesterase; thebutyrylcholinesterase variant Y332S designated SEQ ID NO: 20 exhibits anincreased cocaine hydrolysis activity compared to butyrylcholinesterase;the butyrylcholinesterase variant Y332M designated SEQ ID NO: 22exhibits an increased cocaine hydrolysis activity compared tobutyrylcholinesterase; the butyrylcholinesterase variant Y332Pdesignated SEQ ID NO: 24 exhibits an increased cocaine hydrolysisactivity compared to butyrylcholinesterase; the butyrylcholinesterasevariant V331L designated SEQ ID NO: 26 exhibits an increased cocainehydrolysis activity compared to butyrylcholinesterase; thebutyrylcholinesterase variant F227A designated SEQ ID NO: 28 exhibitsabout a four-fold increased cocaine hydrolysis activity compared tobutyrylcholinesterase; the butyrylcholinesterase variant F227Gdesignated SEQ ID NO: 30 exhibits about a four-fold increased cocainehydrolysis activity compared to butyrylcholinesterase; thebutyrylcholinesterase variant F227S designated SEQ ID NO: 32 exhibitsabout a two-fold increased cocaine hydrolysis activity compared tobutyrylcholinesterase; the butyrylcholinesterase variant F227Pdesignated SEQ ID NO: 34 exhibits about a three-fold increased cocainehydrolysis activity compared to butyrylcholinesterase; thebutyrylcholinesterase variant F227T designated SEQ ID NO: 36 exhibitsabout a two-fold increased cocaine hydrolysis activity compared tobutyrylcholinesterase; the butyrylcholinesterase variant F227Cdesignated SEQ ID NO: 38 exhibits about a two-fold increased cocainehydrolysis activity compared to butyrylcholinesterase; thebutyrylcholinesterase variant F227M designated SEQ ID NO: 40 exhibitsabout a one-and-a-half-fold increased cocaine hydrolysis activitycompared to butyrylcholinesterase; the butyrylcholinesterase variantA199S designated SEQ ID NO: 42 exhibits about a two-and-a-half-foldincreased cocaine hydrolysis activity compared to butyrylcholinesterase;and the butyrylcholinesterase variant A328W/Y332M/S287G/F227A designatedSEQ ID NO: 52, also referred to as AME-359 herein, exhibits about aone-hundred-fold increased cocaine hydrolysis activity compared tobutyrylcholinesterase.

One skilled in the art will appreciate that the exact increase incocaine hydrolysis activity compared to butyrylcholinesterase that isdetected depends on the particular assay chosen. Therefore, while all ofthe butyrylcholinesterase variants of the invention have increasedcocaine hydrolysis activity, the values set forth herein are approximatevalues that can vary if a different assay were performed.

It is understood that minor modifications in the primary amino acidsequence can result in a polypeptide that has a similar, non-identicalsequence, but retains comparable functional or biological activity to abutyrylcholinesterase variant polypeptide of the invention. Thesemodifications can be deliberate, as through site-directed mutagenesis,or may be accidental such as through spontaneous mutation. For example,it is understood that only a portion of the entire primary structure ofa butyrylcholinesterase variant polypeptide can retain the cocainehydrolysis activity of the reference butyrylcholinesterase variantpolypeptide. Such functional fragments of the sequence of abutyrylcholinesterase variant polypeptide of the invention are includedwithin the definition as long as at least one biological function of thebutyrylcholinesterase variant is retained. It is understood that variousmolecules can be attached to a polypeptide of the invention, forexample, other polypeptides, carbohydrates, lipids, or chemicalmoieties.

The term “functional fragment,” when used in reference to abutyrylcholinesterase variant polypeptide of the invention, refers to apolypeptide fragment that is a portion of the butyrylcholinesterasevariant polypeptide, provided that the portion has a biologicalactivity, as described herein, that is characteristic of the referencebutyrylcholinesterase variant polypeptide. The amino acid length of afunctional fragment of a butyrylcholinesterase variant polypeptide ofthe present invention can range from about 5 amino acids up to thefull-length protein sequence of the reference butyrylcholinesterasevariant polypeptide. In certain embodiments, the amino acid lengthsinclude, for example, at least about 10 amino acids, at least about 15,at least about 20, at least about 25, at least about 30, at least about35, at least about 40, at least about 45, at least about 50, at leastabout 75, at least about 100, at least about 150, at least about 200, atleast about 250 or more amino acids in length up to the full-lengthbutyrylcholinesterase variant polypeptide sequence. The functionalfragments can be contiguous amino acid sequences of abutyrylcholinesterase variant polypeptide, including contiguous aminoacid sequence corresponding to the substrate binding domain of thebutyrylcholinesterase variant polypeptide. A functional fragment of abutyrylcholinesterase variant polypeptide of the invention exhibiting afunctional activity can have, for example, at least 8, 10, 15, 20, 30 or40 amino acids, and often has at least 50, 75, 100, 200, 300, 400 ormore amino acids of a polypeptide of the invention, up to the fulllength polypeptide minus one amino acid. The appropriate length andamino acid sequence of a functional fragment of a polypeptide of theinvention can be determined by those skilled in the art, depending onthe intended use of the functional fragment. For example, a functionalfragment of a butyrylcholinesterase variant is intended to refer to aportion of the butyrylcholinesterase variant that still retains some orall of the cocaine hydrolysis activity of the parent polypeptide.

A functional fragment of a butyrylcholinesterase variant polypeptide cancontain active site residues important for the catalytic activity of theenzyme. Regions important for the hydrolysis activity of abutyrylcholinesterase variant polypeptide can be determined or predictedthrough a variety of methods known in the art. Related enzymes such as,for example, acetylcholinesterase and carboxylesterase, that share ahigh degree of sequence similarity and have biochemically similarcatalytic properties can provide information regarding the regionsimportant for catalytic activity of a butyrylcholinesterase variantpolypeptide. For example, structural modeling can reveal the active siteof an enzyme, which is a three-dimensional structure such as a cleft,gorge or crevice formed by amino acid residues generally located apartfrom each other in primary structure. Therefore, a functional fragmentof a butyrylcholinesterase variant polypeptide of the invention canencompass amino acid residues that make up regions of abutyrylcholinesterase enzyme important for cocaine hydrolysis activitysuch as those residues located along the active site gorge.

In addition to structural modeling of a butyrylcholinesterase enzyme,biochemical data can be used to determine or predict regions of abutyrylcholinesterase variant polypeptide important for cocainehydrolysis activity when preparing a functional fragment of abutyrylcholinesterase variant polypeptide of the invention. In thisregard, the characterization of naturally occurringbutyrylcholinesterase enzymes with altered cocaine hydrolysis activitycan be useful for identifying regions important for the catalyticactivity of a butyrylcholinesterase variant polypeptide. Similarly,site-directed mutagenesis studies can provide data regardingcatalytically important amino acid residues as reviewed, for example, inSchwartz et al., Pharmac. Ther. 67: 283-322 (1992), which isincorporated by reference. In particular, a functional fragment of abutyrylcholinesterase variant polypeptide can include the active siteresidues corresponding to amino acid positions 82, 112, 128, 231, 329,332, 430 and 440 of the butyrylcholinesterase shown as SEQ ID NO: 14.Thus, a functional fragment can, for example, be 360 amino acid residuesin length and can include residues 80 to 440 of the referencebutyrylcholinesterase variant polypeptide.

Therefore, a functional fragment of a butyrylcholinesterase variantpolypeptide can encompass an area or region of the amino acid sequenceof butyrylcholinesterase that is determined or predicted to be importantfor cocaine hydrolysis activity. As described above, a region can bedetermined or predicted to be important for cocaine hydrolysis activityby using one or more of structural, biochemical or modeling methods and,as a consequence, is defined by general rather than absolute boundaries.A region can encompass two or more consecutive amino acid positions ofthe amino acid sequence of butyrylcholinesterase that are predicted tobe important for cocaine hydrolysis activity. A region ofbutyrylcholinesterase useful as a functional fragment of abutyrylcholinesterase variant polypeptide for practicing the claimedinvention is no more than about 30 amino acids in length and preferablyis between 2 and 20, between 5 and 15 amino acids in length.

A butyrylcholinesterase variant polypeptide of the invention, or afunctional fragment thereof, can have conservative amino acidsubstitutions as compared with the reference butyrylcholinesterasevariant amino acid sequence. Conservative substitutions of encoded aminoacids include, for example, amino acids that belong within the followinggroups: (1) non-polar amino acids (Gly, Ala, Val, Leu, and Ile); (2)polar neutral amino acids (Cys, Met, Ser, Thr, Asn, and Gln); (3) polaracidic amino acids (Asp and Glu); (4) polar basic amino acids (Lys, Argand His); and (5) aromatic amino acids (Phe, Trp, Tyr, and His).

A butyrylcholinesterase variant polypeptide having the same orsubstantially the same amino acid sequence of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and52, or a functional fragment thereof, also can be chemically modified,provided that the polypeptide retains a biological activity of thereference butyrylcholinesterase variant polypeptide. For example,chemical modification of a butyrylcholinesterase variant polypeptide ofthe invention can include alkylation, acylation, carbamylation andiodination. Moreover, modified polypeptides also can include thosepolypeptides in which free amino groups have been derivatized to form,for example, amine hydrochlorides, p-toluene sulfonyl groups,carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups orformyl groups. Free carboxyl groups can be modified to form salts,methyl and ethyl esters or other types of esters or hydrazides. Freehydroxyl groups can be modified to form O-acyl or O-alkyl, derivatives.The imidazole nitrogen of histidine can be derivatized to formN-im-benzylhistidine. A butyrylcholinesterase variant polypeptide of theinvention also can include a variety of other modifications well knownto those skilled in the art, provided the biological activity of thereference butyrylcholinesterase variant polypeptide remainssubstantially unaffected.

An isolated polypeptide having the same or substantially the same aminoacid sequence of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24,26, 28, 30, 32, 34, 36, 38, 40, 42 or 52, or a functional fragmentthereof, also can be substituted with one or more amino acid analogs ofthe twenty standard amino acids, for example, 4-hydroxyproline,5-hydroxylysine, 3-methylhistidine, homoserine, ornithine orcarboxyglutamate, and can include amino acids that are not linked bypeptide bonds.

A butyrylcholinesterase variant polypeptide having the same orsubstantially the same amino acid sequence of SEQ ID NOS: 2, 4, 6, 8,10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 or52, or a functional fragment thereof, also can contain mimetic portionsthat orient functional groups, which provide a function of abutyrylcholinesterase enzyme. Therefore, mimetics encompass chemicalscontaining chemical moieties that mimic the function of the polypeptide.For example, if a polypeptide contains similarly charged chemicalmoieties having similar functional activity, a mimetic places similarcharged chemical moieties in a similar spatial orientation andconstrained structure so that the chemical function of the chargedmoieties is maintained. Exemplary mimetics are peptidomimetics,peptoids, or other peptide-like polymers such as poly-β-amino acids, andalso non-polymeric compounds upon which functional groups that mimic apeptide are positioned.

A butyrylcholinesterase variant of the invention can be prepared by avariety of methods well known in the art. If desired, random mutagenesiscan be performed to prepare a butyrylcholinesterase variant of theinvention. Alternatively, as disclosed herein, site-directed mutagenesisbased on the information obtained from structural, biochemical andmodeling methods described herein can be performed to target those aminoacids predicted to be important for cocaine hydrolysis activity. Forexample, molecular modeling of cocaine in the active site ofbutyrylcholinesterase can be utilized to predict amino acid alterationsthat allow for higher catalytic efficiency based on a better fit betweenthe enzyme and its substrate. As described herein, residues predicted tobe important for cocaine hydrolysis activity include 8 hydrophobic gorgeresidues and the catalytic triad residues. Furthermore, it is understoodthat amino acid alterations of residues important for the functionalstructure of a butyrylcholinesterase variant, which include the cysteineresidues ⁶⁵Cys-⁹²Cys, ²⁵²Cys-²⁶³Cys, and ⁴⁰⁰Cys-⁵¹⁹Cys involved inintrachain disulfide bonds are generally not altered in the preparationof a butyrylcholinesterase variant that has cocaine hydrolysis activity.

Following mutagenesis of butyrylcholinesterase or abutyrylcholinesterase variant expression, purification and functionalcharacterization of the butyrylcholinesterase variant can be performedby methods well known in the art. As disclosed below, abutyrylcholinesterase variant can be expressed in an appropriate hostcell line and subsequently purified and characterized for cocainehydrolysis activity. Butyrylcholinesterase variants characterized ashaving significantly increased cocaine hydrolysis activity cansubsequently be used in the methods of hydrolyzing a cocaine-basedsubstrate as well as the methods of treating a cocaine-induced conditiondescribed below.

A butyrylcholinesterase variant of the invention exhibits cocainehydrolysis activity. As disclosed herein, a butyrylcholinesterasevariant of the invention can have increased cocaine hydrolysis activitycompared to butyrylcholinesterase and can be used to treat acocaine-induced condition. A polypeptide having minor modificationscompared to a butyrylcholinesterase variant of the invention isencompassed by the invention so long as equivalent cocaine hydrolysisactivity is retained. In addition, functional fragments of abutyrylcholinesterase variant that still retain some or all of thecocaine hydrolysis activity of the parent butyrylcholinesterase variantare similarly included in the invention. Similarly, functional fragmentsof nucleic acids, which encode functional fragments of abutyrylcholinesterase variant of the invention are similarly encompassedby the invention.

A functional fragment of a butyrylcholinesterase variant of theinvention can be prepared by recombinant methods involving expression ofa nucleic acid molecule encoding the butyrylcholinesterase variant orfunctional fragment thereof, followed by isolation of the variant orfunctional fragment thereof by routine biochemical methods describedherein. It is understood that functional fragments also can be preparedby enzymatic or chemical cleavage of the full lengthbutyrylcholinesterase variant. Methods for enzymatic and chemicalcleavage and for purification of the resultant peptide fragments arewell known in the art (see, for example, Deutscher, Methods inEnzymology, Vol. 182, “Guide to Protein Purification,” San Diego:Academic Press, Inc. (1990), which is incorporated herein by reference).

Furthermore, functional fragments of a butyrylcholinesterase variant canbe produced by chemical synthesis. If desired, such molecules can bemodified to include D-stereoisomers, non-naturally occurring aminoacids, and amino acid analogs and mimetics in order to optimize theirfunctional activity, stability or bioavailability.) Examples of modifiedamino acids and their uses are presented in Sawyer, Peptide Based DrugDesign, ACS, Washington (1995) and Gross and Meienhofer, The Peptides:Analysis, Synthesis, Biology, Academic Press, Inc., New York (1983),both of which are incorporated herein by reference.

If desired, random segments of a butyrylcholinesterase variant can beprepared and tested in the assays described herein. A fragment havingany desired boundaries and modifications compared to the amino acidsequence of the reference butyrylcholinesterase variant of the inventioncan be prepared. Alternatively, available information obtained by thestructural, biochemical and modeling methods described herein can beused to prepare only those fragments of a butyrylcholinesterase variantthat are likely to retain the cocaine hydrolysis activity of the parentvariant. As described herein, residues predicted to be important forcocaine hydrolysis activity include 8 hydrophobic gorge residues and thecatalytic triad residues. Furthermore, residues important for thefunctional structure of a butyrylcholinesterase variant include thecysteine residues ⁶⁵Cys-⁹²Cys, ²⁵²Cys-263Cys, and ⁴⁰⁰Cys⁻⁵¹⁹Cys involvedin intrachain disulfide bonds. Therefore, a functional fragment can be atruncated form, region or segment of the reference butyrylcholinesterasevariant designed to possess most or all of the residues critical forcocaine hydrolysis activity or functional structure so as to retainequivalent cocaine hydrolysis activity. Similarly, a functional fragmentcan include non-peptidic structural elements that serve to mimicstructurally or functionally important residues of the referencevariant. Also included as butyrylcholinesterase variants of theinvention are fusion proteins that result from linking abutyrylcholinesterase variant or functional fragment thereof to aheterologous protein, such as a therapeutic protein, as well as fusionconstructs of nucleic acids encoding such fusion proteins. Fragments ofnucleic acids that can hybridize to a butyrylcholinesterase variant orfunctional fragment thereof are useful, for example, as hybridizationprobes and are also encompassed by the claimed invention.

Thus, the invention provides twenty-one butyrylcholinesterase variantsencompassing the same or substantially the same amino acid sequencesshown as SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28,30, 32, 34, 36, 38, 40, 42 and 52, and functional fragments thereof. Asdescribed herein, each of the invention butyrylcholinesterase variantsexhibits about an increased cocaine hydrolysis activity compared tobutyrylcholinesterase.

The invention also provides twenty-one nucleic acids shown as SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35,37, 39, 41 and 51, respectively, and fragments thereof, which encode thebutyrylcholinesterase variants encompassing the same or substantiallythe same amino acid sequences shown as SEQ ID NOS: 2, 4, 6, 8, 10, 12,14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 52,respectively. Thus, the present invention provides nucleic acids thatencode a butyrylcholinesterase variant encompassing the same orsubstantially the same amino acid sequences shown as SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42and 52.

It is understood that a nucleic acid molecule of the invention or afragment thereof includes sequences having one or more additions,deletions or substitutions with respect to the reference sequence, solong as the nucleic acid molecule retains its ability to selectivelyhybridize with the subject nucleic acid molecule under moderatelystringent conditions, or highly stringent conditions. Moderatelystringent conditions are hybridization conditions equivalent tohybridization of filter-bound nucleic acid in 50% formamide, 5×Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C., followed by washing in0.2×SSPE, 0.2% SDS, at 50°. Highly stringent conditions refers toconditions equivalent to hybridization of filter-bound nucleic acid in50% formamide, 5× Denhardt's solution, 5×SSPE, 0.2% SDS at 42° C.,followed by washing in 0.2×SSPE, 0.2% SDS, at 65°. Other suitablemoderately stringent and highly stringent hybridization buffers andconditions are well known to those of skill in the art and aredescribed, for example, in Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd ed., Cold Spring Harbor Press, Plainview, N.Y.(1989); and Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, New York (2000). Thus, it is not necessary that twonucleic acids exhibit sequence identity to be substantiallycomplementary, only that they can specifically hybridize or be made tospecifically hybridize without detectible cross reactivity with othersimilar sequences.

In general, a nucleic acid molecule that has substantially the samenucleotide sequence as a reference sequence will have greater than about60% identity, such as greater than about 65%, 70%, 75% identity with thereference sequence, such as greater than about 80%, 85%, 90%, 95%, 97%or 99% identity to the reference sequence over the length of the twosequences being compared. Identity of any two nucleic acid sequences canbe determined by those skilled in the art based, for example, on a BLAST2.0 computer alignment, using default parameters. BLAST 2.0 searching isavailable at ncbi.nlm.nih.gov/gorf/bl2.html., as described by Tatiana etal., FEMS Microbiol Lett. 174:247-250 (1999).

As used herein, the term “fragment” when used in reference to a nucleicacid encoding the claimed polypeptides is intended to mean a nucleicacid having substantially the same sequence as a portion of a nucleicacid encoding a polypeptide of the invention or segments thereof. Thenucleic acid fragment is sufficient in length and sequence toselectively hybridize to a butyrylcholinesterase variant encodingnucleic acid or a nucleotide sequence that is complementary to abutyrylcholinesterase variant encoding nucleic acid. Therefore, fragmentis intended to include primers for sequencing and polymerase chainreaction (PCR) as well as probes for nucleic acid blot or solutionhybridization.

Similarly, the term “functional fragment” when used in reference to anucleic acid encoding a butyrylcholinesterase or butyrylcholinesterasevariant is intended to refer to a portion of the nucleic acid thatencodes a portion of the butyrylcholinesterase variant that stillretains some or all of the cocaine hydrolysis activity of the referencevariant polypeptide. A functional fragment of a polypeptide of theinvention exhibiting a functional activity can have, for example, atleast 6 contiguous amino acid residues from the polypeptide, at least 8,10, 15, 20, 30 or 40 amino acids, and often has at least 50, 75, 100,200, 300, 400 or more amino acids of a polypeptide of the invention, upto the full length polypeptide minus one amino acid.

As used herein, the term “cocaine hydrolysis activity,” is intended torefer to the catalytic action of a butyrylcholinesterase orbutyrylcholinesterase variant as measured by the rate of cocainehydrolysis into the metabolites.

As used herein, the term “effective amount” is intended to mean anamount of a butyrylcholinesterase variant of the invention that canreduce the cocaine-toxicity or the severity of a cocaine-inducedcondition. Reduction in severity includes, for example, an arrest or adecrease in symptoms, physiological indicators, biochemical markers ormetabolic indicators. Symptoms of cocaine overdose include, for example,cardiac arrythmias, seizures and hypertensive crises. A reduction inseverity also includes a delay in the onset of symptoms. As used herein,the term “treating” is intended to mean causing a reduction in theseverity of a cocaine-induced condition.

As used herein, the term “cocaine-based substrate” refers to (−)-cocaineor any molecule sufficiently similar to (−)-cocaine in structure to behydrolyzed by butyrylcholinesterase or a butyrylcholinesterase variantincluding, for example, (+)-cocaine, acetylcholine, butyrylthiocholine,benzoylcocaine and norcocaine.

The nucleic acid shown as SEQ ID NO: 1, or fragment thereof, encodes abutyrylcholinesterase variant encompassing the same or substantially thesame amino acid sequence shown as SEQ ID NO: 2. As shown in Table 1, thenucleic acid shown as SEQ ID: 1 differs from the nucleic acid encodinghuman butyrylcholinesterase shown in FIG. 3 at the codon positionsencoding amino acid residues 328 and 332, respectively. In the humanbutyrylcholinesterase (SEQ ID NO: 13) the codons gct and tat encodeAlanine at amino acid position 328 and Tyrosine at amino acid position332, respectively. In contrast, in the nucleic acid encoding theA328W/Y332M butyrylcholinesterase variant designated SEQ ID NO: 2, thecodons tgg and atg encode Tryptophan at amino acid position 328 andMethionine at amino acid position 332, respectively.

The invention provides a further nucleic acid shown as SEQ ID NO: 3, orfragment thereof, encodes a butyrylcholinesterase variant encompassingthe same or substantially the same amino acid sequence shown as SEQ IDNO: 4. As shown in Table 1, the nucleic acid shown as SEQ ID: 3 differsfrom the nucleic acid encoding human butyrylcholinesterase shown in FIG.3 and designated SEQ ID NO: 13, at the codons encoding amino acidresidues 328 and 332. In the human butyrylcholinesterase (SEQ ID NO: 13)the codons gct and tat encode Alanine at amino acid position 328 andTyrosine at amino acid position 332. In contrast, in the nucleic acidencoding the A328W/Y332P butyrylcholinesterase variant designated SEQ IDNO: 4, the codons tgg and cca encode Tryptophan at amino acid position328 and Proline at amino acid position 332.

The invention provides a further nucleic acid shown as SEQ ID NO: 5, orfragment thereof, encodes a butyrylcholinesterase variant encompassingthe same or substantially the same amino acid sequence shown as SEQ IDNO: 6. As shown in Table 1, the nucleic acid shown as SEQ ID: 5 differsfrom the nucleic acid encoding human butyrylcholinesterase shown in FIG.3 and designated SEQ ID NO: 43, at the codon positions encoding aminoacid residues 328 and 331. In the human butyrylcholinesterase (SEQ IDNO: 43) the codons gct and gtc encode Alanine at amino acid position 328and Valine at amino acid position 331. In contrast, in the nucleic acidencoding the A328W/V331L butyrylcholinesterase variant designated SEQ IDNO: 6, the corresponding codons encode Tryptophan at amino acid position328 and Leucine at amino acid position 331.

The invention provides a further nucleic acid shown as SEQ ID NO: 7, orfragment thereof, encodes a butyrylcholinesterase variant encompassingthe same or substantially the same amino acid sequence shown as SEQ IDNO: 8. As shown in Table 1, the nucleic acid shown as SEQ ID: 7 differsfrom the nucleic acid encoding human butyrylcholinesterase shown in FIG.3 and designated SEQ ID NO: 43 at the codon positions encoding aminoacid residues 328 and 332. In the human butyrylcholinesterase (SEQ IDNO: 43) the codons gct and tat encode Alanine at amino acid position 328and Tyrosine at amino acid position 332. In contrast, in the nucleicacid encoding the A328W/Y332S butyrylcholinesterase variant designatedSEQ ID NO: 8, the codons tgg and tcg encode Tryptophan at amino acidposition 328 and Serine at amino acid position 332.

The invention provides a further nucleic acid shown as SEQ ID NO: 9, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 10. As shown in Table 1, the nucleic acid shown asSEQ ID: 9 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon positions encoding amino acid residues 199, 227, 287, 332 and328. In the human butyrylcholinesterase (SEQ ID NO: 43) the codons gca,ttt, tca, gct and tat encode Alanine at amino acid position 199,Phenylalanine at amino acid position 227, Serine at amino acid position287, Alanine at amino acid position 328 and Tyrosine at amino acidposition 332. In contrast, in the nucleic acid encoding theA328W/Y332M/S287G/F227A/A199S butyrylcholinesterase variant designatedSEQ ID NO: 10, the codons tca, gcg, ggt, tgg and atg encode Serine atamino acid position 199, Alanine at amino acid position 227, Glycine atamino acid position 287, Tryptophan at amino acid position 328 andMethionine at amino acid position 332, respectively.

The invention provides a further nucleic acid shown as SEQ ID NO: 11, orfragment thereof, encodes a butyrylcholinesterase variant encompassingthe same or substantially the same amino acid sequence shown as SEQ IDNO: 12. As shown in Table 1, the nucleic acid shown as SEQ ID: 11differs from the nucleic acid encoding human butyrylcholinesterase shownin FIG. 3 and designated SEQ ID NO: 43, at the codon positions encodingamino acid residues 199, 227, 287 and 328, respectively. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codons gca, ttt, tca and gctencode Alanine at amino acid position 199, Phenylalanine at amino acidposition 227, Serine at amino acid position 287, and Alanine at aminoacid position 328, respectively. In contrast, in the nucleic acidencoding the A328W/S287G/F227A/A199S butyrylcholinesterase variantdesignated SEQ ID NO: 12, the codons tca, gcg, ggt and tgg encode Serineat amino acid position 199, Alanine at amino acid position 227, Glycineat amino acid position 287 and Tryptophan at amino acid position 328,respectively.

The invention provides a further nucleic acid shown as SEQ ID NO: 13, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 14. As shown in Table 1, the nucleic acid shown asSEQ ID: 13 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon positions encoding amino acid residues 199, 287 and 328,respectively. In the human butyrylcholinesterase (SEQ ID NO: 43) thecodons gca, tca and gct encode Alanine at amino acid position 199,Serine at amino acid position 287 and Alanine at amino acid position328, respectively. In contrast, in the nucleic acid encoding theA328W/S287G/A199S butyrylcholinesterase variant designated SEQ ID NO:14, the codons tca, ggt and tgg, encode Serine at amino acid position199, Glycine at amino acid position 287 and Tryptophan at amino acidposition 328, respectively.

The invention provides a further nucleic acid shown as SEQ ID NO: 15, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 16. As shown in Table 1, the nucleic acid shown asSEQ ID: 15 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon positions encoding amino acid residues 227, 287 and 328,respectively. In the human butyrylcholinesterase (SEQ ID NO: 43) thecodons ttt, tca, gct encode Phenylalanine at amino acid position 227,Serine at amino acid position 287 and Alanine at amino acid position328, respectively. In contrast, in the nucleic acid encoding theA328W/S287G/F227A butyrylcholinesterase variant designated SEQ ID NO:16, the codons gcg, ggt and tgg encode Alanine at amino acid position227, Glycine at amino acid position 287 and Tryptophan at amino acidposition 328, respectively.

The invention provides a further nucleic acid shown as SEQ ID NO: 17, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 18. As shown in Table 1, the nucleic acid shown asSEQ ID: 17 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon positions encoding amino acid residues 227 and 328,respectively. In the human butyrylcholinesterase (SEQ ID NO: 43) thecodons ttt and gct at nucleotide encode Phenylalanine at amino acidposition 227 and Alanine at amino acid position 328, respectively. Incontrast, in the nucleic acid encoding the A328W/F227Abutyrylcholinesterase variant designated SEQ ID NO: 18, the codons gcgand tgg encode Alanine at amino acid position 227 and Tryptophan atamino acid position 328, respectively.

The invention provides a further nucleic acid shown as SEQ ID NO: 19, orfragment thereof, which encodes a butyrylcholinesterase variantcomprising substantially the same amino acid sequence shown as SEQ IDNO: 20. As shown in Table 1, the nucleic acid shown as SEQ ID: 19differs from the nucleic acid encoding human butyrylcholinesterase shownin FIG. 3 and designated SEQ ID NO: 43, at the codon position encodingamino acid residue 332. In the human butyrylcholinesterase (SEQ ID NO:43) the codon tat encodes Tyrosine at amino acid position 332. Incontrast, in the nucleic acid encoding the Y332S butyrylcholinesterasevariant designated SEQ ID NO: 20, the codon tcg encodes Serine at aminoacid position 332.

The invention provides a further nucleic acid shown as SEQ ID NO: 21, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 22. As shown in Table 1, the nucleic acid shown asSEQ ID: 21 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 332. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon tat encodes Tyrosine atamino acid position 332. In contrast, in the nucleic acid encoding theY332M butyrylcholinesterase variant designated SEQ ID NO: 22, the codonatg encodes Methionine at amino acid position 332.

The invention provides a further nucleic acid shown as SEQ ID NO: 23, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 24. As shown in Table 1, the nucleic acid shown asSEQ ID: 23 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 332. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon tat encodes Tyrosine atamino acid position 332. In contrast, in the nucleic acid encoding theY332P butyrylcholinesterase variant designated SEQ ID NO: 24, the codoncca encodes Proline at amino acid position 332.

The invention provides a further nucleic acid shown as SEQ ID NO: 25, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 26. As shown in Table 1, the nucleic acid shown asSEQ ID: 25 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 331. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon gtc encodes Valine atamino acid position 331. In contrast, in the nucleic acid encoding theV331L butyrylcholinesterase variant designated SEQ ID NO: 26, the codonttg encodes Leucine at amino acid position 331.

The invention provides a further nucleic acid shown as SEQ ID NO: 27, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 28. As shown in Table 1, the nucleic acid shown asSEQ ID: 27 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 227. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon ttt encodesPhenylalanine at amino acid position 227. In contrast, in the nucleicacid encoding the F227A butyrylcholinesterase variant designated SEQ IDNO: 28, the codon gcg encodes Alanine at amino acid position 227.

The invention provides a further nucleic acid shown as SEQ ID NO: 29, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 30. As shown in Table 1, the nucleic acid shown asSEQ ID: 29 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 227. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon ttt encodesPhenylalanine at amino acid position 227. In contrast, in the nucleicacid encoding the F227G butyrylcholinesterase variant designated SEQ IDNO: 30, the codon ggg encodes Glycine at amino acid position 227.

The invention provides a further nucleic acid shown as SEQ ID NO: 31, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 32. As shown in Table 1, the nucleic acid shown asSEQ ID: 31 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residues 227. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon ttt encodesPhenylalanine at amino acid position 227. In contrast, in the nucleicacid encoding the F227S butyrylcholinesterase variant designated SEQ IDNO: 32, the codon agt encodes Serine at amino acid position 227.

The invention provides a further nucleic acid shown as SEQ ID NO: 33, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 34. As shown in Table 1, the nucleic acid shown asSEQ ID: 33 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residues 227. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon ttt encodesPhenylalanine at amino acid position 227. In contrast, in the nucleicacid encoding the F227P butyrylcholinesterase variant designated SEQ IDNO: 34, the codon ccg encodes Proline at amino acid position 227.

The invention provides a further nucleic acid shown as SEQ ID NO: 35, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 36. As shown in Table 1, the nucleic acid shown asSEQ ID: 35 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 227. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon ttt encodesPhenylalanine at amino acid position 227. In contrast, in the nucleicacid encoding the F227T butyrylcholinesterase variant designated SEQ IDNO: 36, the codon act encodes Threonine at amino acid position 227.

The invention provides a further nucleic acid shown as SEQ ID NO: 37, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 38. As shown in Table 1, the nucleic acid shown asSEQ ID: 37 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 227. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon ttt encodesPhenylalanine at amino acid position 227. In contrast, in the nucleicacid encoding the F227C butyrylcholinesterase variant designated SEQ IDNO: 38, the codon tgt encodes Cysteine at amino acid position 227.

The invention provides a further nucleic acid shown as SEQ ID NO: 39, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 40. As shown in Table 1, the nucleic acid shown asSEQ ID: 39 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 227. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon ttt encodesPhenylalanine at amino acid position 227. In contrast, in the nucleicacid encoding the F227M butyrylcholinesterase variant designated SEQ IDNO: 40, the codon atg encodes Methionine at amino acid position 227.

The invention provides a further nucleic acid shown as SEQ ID NO: 41, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 42. As shown in Table 1, the nucleic acid shown asSEQ ID: 41 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon position encoding amino acid residue 199. In the humanbutyrylcholinesterase (SEQ ID NO: 43) the codon gca encodes Alanine atamino acid position 199. In contrast, in the nucleic acid encoding theA199S butyrylcholinesterase variant designated SEQ ID NO: 42, the codontca encodes Serine at amino acid position 199.

The invention provides a further nucleic acid shown as SEQ ID NO: 51, orfragment thereof, which encodes a butyrylcholinesterase variantencompassing the same or substantially the same amino acid sequenceshown as SEQ ID NO: 52. As shown in Table 1, the nucleic acid shown asSEQ ID: 52 differs from the nucleic acid encoding humanbutyrylcholinesterase shown in FIG. 3 and designated SEQ ID NO: 43, atthe codon positions encoding amino acid residues 227, 287, 332 and 328.In the human butyrylcholinesterase (SEQ ID NO: 43) the codons ttt, tca,gct and tat encode Alanine at amino acid position 199, Phenylalanine atamino acid position 227, Serine at amino acid position 287, Alanine atamino acid position 328 and Tyrosine at amino acid position 332. Incontrast, in the nucleic acid encoding the A328W/Y332M/S287G/F227Abutyrylcholinesterase variant designated SEQ ID NO: 52, the codons gcg,ggt, tgg and atg encode Alanine at amino acid position 227, Glycine atamino acid position 287, Tryptophan at amino acid position 328 andMethionine at amino acid position 332, respectively.

TABLE 1 Nucleotide Sequences Corresponding to Amino Acid Positions 199,227, 287, 328, 331 and 332. Codon sequences that differ from humanbutyrylcholinesterase (SEQ ID NO: 43) are set forth below. SEQ ID NO na(aa) 199 227 287 328 331 332 Human BchE 43 (44) gca ttt tca gct gtc tatA328W/Y332M  1 (2) tgg atg A328W/Y332P  3 (4) tgg cca A328W/V331L  5 (6)tgg ttg A328W/Y332S  7 (8) tgg tcg A328W/Y332M/  9 (10) tca gcg ggt tggatg S287G/F227A/ A199S A328W/S287G/ 11(12) tca gcg ggt tgg F227A/A199SA328W/S287G/ 13 (14) tca ggt tgg A199S A328W/S287G/ 15 (16) gcg ggt tggF227A A328W/F227A 17 (18) gcg tgg Y332S 19 (20) tcg Y332M 21 (22) atgY332P 23 (24) cca V331L 25 (26) ttg F227A 27 (28) gcg F227G 29 (30) gggF227S 31 (32) agt F227P 33 (34) ccg F227T 35 (36) act F227C 37 (38) tgtF227M 39 (40) atg A199S 41 (42) tca A328W/Y332M/ 51 (52) gcg ggt tgg atgS287G/F227A

A butyrylcholinesterase variant can be obtained by screening a libraryor collection of molecules. A library can contain a few or a largenumber of different molecules, varying from as small as 2 molecules toas large as 10¹³ or more molecules. Therefore, a library can range insize from 2 to 10, 10 to 10², 10² to 10³, 10³ to 10⁵, 10⁵ to 10⁸, 10⁸ to10¹⁰ or 10¹⁰ to 10¹³ molecules. The molecules making up a library can benucleic acid molecules such as an RNA, a cDNA or an oligonucleotide; apeptide or polypeptide including a variant or modified peptide or apeptide containing one or more amino acid analogs. In addition, themolecules making up a library can be peptide-like molecules, referred toherein as peptidomimetics, which mimic the activity of a peptide; or apolypeptide such as an enzyme or a fragment thereof. Moreover, a librarycan be diverse or redundant depending on the intent and needs of theuser. Those skilled in the art will know the size and diversity of alibrary suitable for obtaining a butyrylcholinesterase variantpolypeptide.

A library that is sufficiently diverse to contain abutyrylcholinesterase variant with enhanced cocaine hydrolysis activitycan be prepared by a variety of methods well known in the art. Forexample, a library of butyrylcholinesterase variants can be preparedthat contains each of the 19 amino acids not found in the referencebutyrylcholinesterase at each of the approximately 573 amino acidpositions and screening the resultant variant library forbutyrylcholinesterase variants with enhanced cocaine hydrolysisactivity.

Alternatively, a butyrylcholinesterase variant polypeptide can beobtained from focused library prepared utilizing the structural,biochemical and modeling information relating to butyrylcholinesteraseas described herein. It is understood that any information relevant tothe determination or prediction of residues or regions important for thecocaine hydrolysis activity or structural function ofbutyrylcholinesterase can be useful in the design of a focused libraryof butyrylcholinesterase variants. Thus, the butyrylcholinesterasevariants can be focused to contain amino acid alterations at amino acidpositions located in regions determined or predicted to be important forcocaine hydrolysis activity. A focused library of butyrylcholinesterasevariants can be screened in order to identify a butyrylcholinesterasevariant with enhanced cocaine hydrolysis activity by targeting aminoacid alterations to regions determined or predicted to be important forcocaine hydrolysis activity.

Regions important for the cocaine hydrolysis activity ofbutyrylcholinesterase can be determined or predicted. Related enzymessuch as, for example, acetylcholinesterase and carboxylesterase, thatshare a high degree of sequence similarity and have biochemicallysimilar catalytic properties can provide information regarding theregions important for catalytic activity of butyrylcholinesterase. Forexample, structural modeling can reveal the active site of an enzyme,which is a three-dimensional structure such as a cleft, gorge or creviceformed by amino acid residues generally located apart from each other inprimary structure. Therefore, amino acid residues that make up regionsof butyrylcholinesterase important for cocaine hydrolysis activity caninclude residues located along the active site gorge. For a descriptionof structural modeling of butyrylcholinesterase, see for example, Harelet al., Proc. Nat. Acad. Sci. USA 89: 10827-10831 (1992) and Soreq etal., Trends Biochem. Sci. 17(9): 353-358 (1992), which are incorporatedherein by reference.

In addition to structural modeling of butyrylcholinesterase, biochemicaldata can be used to determine or predict regions ofbutyrylcholinesterase important for cocaine hydrolysis activity. In thisregard, the characterization of naturally occurringbutyrylcholinesterase variants with altered cocaine hydrolysis activityis useful for identifying regions important for the catalytic activityof butyrylcholinesterase. Similarly, site-directed mutagenesis studiescan provide data regarding catalytically important amino acid residuesas reviewed, for example, in Schwartz et al., Pharmac. Ther. 67: 283-322(1992), which is incorporated by reference.

To prepare a butyrylcholinesterase variant having enhanced cocainehydrolysis activity, distinct types of information can be used alone orcombined to determine or predict a region of an amino acid sequence or aspecific amino acid residue of butyrylcholinesterase important forcocaine hydrolysis activity. For example, information based onstructural modeling and biochemical data is combined to determine aregion of an amino acid sequence or a specific amino acid residue ofbutyrylcholinesterase important for cocaine hydrolysis activity. Becauseinformation obtained by a variety of methods can be combined to predictthe catalytically active regions, one skilled in the art will appreciatethat the regions themselves represent approximations rather than strictconfines. As a result, a butyrylcholinesterase variant can have aminoacid alterations outside of the regions determined or predicted to beimportant for cocaine hydrolysis activity. Similarly, abutyrylcholinesterase variant of the invention can have amino acidalterations outside of the regions determined or predicted to beimportant for cocaine hydrolysis activity. Furthermore, abutyrylcholinesterase variant of the invention can have any othermodification that does not significantly change its cocaine hydrolysisactivity. It is further understood that the number of regions determinedor predicted to be important for cocaine hydrolysis activity can varybased on the predictive methods used.

Once a number of regions or specific residues have been identified byany method appropriate for determination of regions or specific aminoacid residues important for cocaine hydrolysis, each region or specificpositions can be randomized across some or all amino acid positions tocreate a library of variants containing the wild-type amino acid plusone or more of the other nineteen naturally occurring amino acids at oneor more positions within each of the regions. As summarized in Table 2,regions of an amino acid sequence of butyrylcholinesterase important forcocaine hydrolysis can include, for example, amino acid residues 68through 82, 110 through 121, 194 through 201, 224 through 234, 277through 289, 327 through 332, and 429 through 442 corresponding to thehuman butyrylcholinesterase designated SEQ ID NO: 44.

Methods for preparing libraries containing diverse populations ofvarious types of molecules such as peptides, peptoids andpeptidomimetics are well known in the art (see, for example, Ecker andCrooke, Biotechnology 13:351-360 (1995), and Blondelle et al., TrendsAnal. Chem. 14:83-92 (1995), and the references cited therein, each ofwhich is incorporated herein by reference; see, also, Goodman and Ro,Peptidomimetics for Drug Design, in “Burger's Medicinal Chemistry andDrug Discovery” Vol. 1 (ed. M. E. Wolff; John Wiley & Sons 1995), pages803-861, and Gordon et al. J. Med. Chem. 37:1385-1401 (1994), each ofwhich is incorporated herein by reference). Where a molecule is apeptide, protein or fragment thereof, the molecule can be produced invitro directly or can be expressed from a nucleic acid, which can beproduced in vitro. Methods of synthetic peptide chemistry are well knownin the art.

A butyrylcholinesterase variant of the invention also can be produced,for example, by constructing and subsequently screening a nucleic acidexpression library encoding butyrylcholinesterase variants. Methods forproducing such libraries are well known in the art (see, for example,Sambrook et al., supra, 1989). A library of nucleic acids can becomposed of DNA, RNA or analogs thereof. A library containing RNAmolecules can be constructed, for example, by synthesizing the RNAmolecules chemically.

A nucleic acid encoding a butyrylcholinesterase variant can be obtainedby any means desired by the user. Those skilled in the art will knowwhat methods can be used to obtain a nucleic acid encodingbutyrylcholinesterase variant of the invention. For example, abutyrylcholinesterase variant can be generated by mutagenesis of nucleicacids encoding butyrylcholinesterase using methods well known to thoseskilled in the art (Molecular Cloning: A Laboratory Manual, Sambrook etal., supra, 1989). A butyrylcholinesterase variant of the invention canbe obtained from a library of nucleic acids that is randomized to besufficiently diverse to contain nucleic acids encoding every possiblenaturally occurring amino acid at each amino acid position ofbutyrylcholinesterase. Alternatively, a butyrylcholinesterase variant ofthe invention can be obtained from a library of nucleic acids such thatit contains a desired amino acid at a predetermined position predictedor determined to be important for cocaine hydrolysis activity.

One or more mutations can be introduced into a nucleic acid moleculeencoding a butyrylcholinesterase variant to yield a modified nucleicacid molecule using, for example, site-directed mutagenesis (see Wu(Ed.), Meth. In Enzymol. Vol. 217, San Diego: Academic Press (1993);Higuchi, “Recombinant PCR” in Innis et al. (Ed.), PCR Protocols, SanDiego: Academic Press, Inc. (1990), each of which is incorporated hereinby reference). Such mutagenesis can be used to introduce a specific,desired amino acid alteration.

The efficient synthesis and expression of libraries ofbutyrylcholinesterase variants using oligonucleotide-directedmutagenesis can be accomplished as previously described by Wu et al.,Proc. Natl. Acad. Sci. USA, 95:6037-6042 (1998); Wu et al., J. Mol.Biol., 294:151-162 (1999); and Kunkel, Proc. Natl. Acad. Sci. USA,82:488-492 (1985), which are incorporated herein by reference.Oligonucleotide-directed mutagenesis is a well-known and efficientprocedure for systematically introducing mutations, independent of theirphenotype and is, therefore, ideally suited for directed evolutionapproaches to protein engineering. To perform oligonucleotide-directedmutagenesis a library of nucleic acids encoding the desired mutations ishybridized to single-stranded uracil-containing template of thewild-type sequence. The methodology is flexible, permitting precisemutations to be introduced without the use of restriction enzymes, andis relatively inexpensive if oligonucleotides are synthesized usingcodon-based mutagenesis.

Codon-based synthesis or mutagenesis represents one method well known inthe art for avoiding genetic redundancy while rapidly and efficientlyproducing a large number of alterations in a known amino acid sequenceor for generating a diverse population of random sequences. This methodis the subject matter of U.S. Pat. Nos. 5,264,563 and 5,523,388 and isalso described in Glaser et al. J. Immunology 149:3903-3913 (1992).Briefly, coupling reactions for the randomization of, for example, alltwenty codons which specify the amino acids of the genetic code areperformed in separate reaction vessels and randomization for aparticular codon position occurs by mixing the products of each of thereaction vessels. Following mixing, the randomized reaction productscorresponding to codons encoding an equal mixture of all twenty aminoacids are then divided into separate reaction vessels for the synthesisof each randomized codon at the next position. If desired, equalfrequencies of all twenty amino acids can be achieved with twentyvessels that contain equal portions of the twenty codons. Thus, it ispossible to utilize this method to generate random libraries of theentire sequence of butyrylcholinesterase or focused libraries of theregions or specific positions determined or predicted to be importantfor cocaine hydrolysis activity.

Variations to the above synthesis method also exist and include, forexample, the synthesis of predetermined codons at desired positions andthe biased synthesis of a predetermined sequence at one or more codonpositions as described by Wu et al, supra, 1998. Biased synthesisinvolves the use of two reaction vessels where the predetermined orparent codon is synthesized in one vessel and the random codon sequenceis synthesized in the second vessel. The second vessel can be dividedinto multiple reaction vessels such as that described above for thesynthesis of codons specifying totally random amino acids at aparticular position. Alternatively, a population of degenerate codonscan be synthesized in the second reaction vessel such as through thecoupling of NNG/T nucleotides or NNX/X where N is a mixture of all fournucleotides. Following synthesis of the predetermined and random codons,the reaction products in each of the two reaction vessels are mixed andthen redivided into an additional two vessels for synthesis at the nextcodon position.

A modification to the above-described codon-based synthesis forproducing a diverse number of variant sequences can similarly beemployed for the production of the libraries of butyrylcholinesterasevariants described herein. This modification is based on the two vesselmethod described above which biases synthesis toward the parent sequenceand allows the user to separate the variants into populations containinga specified number of codon positions that have random codon changes.

Briefly, this synthesis is performed by continuing to divide thereaction vessels after the synthesis of each codon position into two newvessels. After the division, the reaction products from each consecutivepair of reaction vessels, starting with the second vessel, is mixed.This mixing brings together the reaction products having the same numberof codon positions with random changes. Synthesis proceeds by thendividing the products of the first and last vessel and the newly mixedproducts from each consecutive pair of reaction vessels and redividinginto two new vessels. In one of the new vessels, the parent codon issynthesized and in the second vessel, the random codon is synthesized.For example, synthesis at the first codon position entails synthesis ofthe parent codon in one reaction vessel and synthesis of a random codonin the second reaction vessel. For synthesis at the second codonposition, each of the first two reaction vessels is divided into twovessels yielding two pairs of vessels. For each pair, a parent codon issynthesized in one of the vessels and a random codon is synthesized inthe second vessel. When arranged linearly, the reaction products in thesecond and third vessels are mixed to bring together those productshaving random codon sequences at single codon positions. This mixingalso reduces the product populations to three, which are the startingpopulations for the next round of synthesis. Similarly, for the third,fourth and each remaining position, each reaction product population forthe preceding position are divided and a parent and random codonsynthesized.

Following the above modification of codon-based synthesis, populationscontaining random codon changes at one, two, three and four positions aswell as others can be conveniently separated out and used based on theneed of the individual. Moreover, this synthesis scheme also allowsenrichment of the populations for the randomized sequences over theparent sequence since the vessel containing only the parent sequencesynthesis is similarly separated out from the random codon synthesis.This method can be used to synthesize a library of nucleic acidsencoding butyrylcholinesterase variants having amino acid alterations inone or more regions of butyrylcholinesterase predicted to be importantfor cocaine hydrolysis activity.

Alternatively, a library of nucleic acids encoding butyrylcholinesterasevariants can also be generated using gene shuffling. Gene shuffling orDNA shuffling is a method for directed evolution that generatesdiversity by recombination (see, for example, Stemmer, Proc. Natl. Acad.Sci. USA 91:10747-10751 (1994); Stemmer, Nature 370:389-391 (1994);Crameri et al., Nature 391:288-291 (1998); Stemmer et al., U.S. Pat. No.5,830,721, issued Nov. 3, 1998). Gene shuffling or DNA shuffling is amethod using in vitro homologous recombination of pools of selectedmutant genes. For example, a pool of point mutants of a particular genecan be used. The genes are randomly fragmented, for example, usingDNase, and reassembled by PCR. If desired, DNA shuffling can be carriedout using homologous genes from different organisms to generatediversity (Crameri et al., supra, 1998). The fragmentation andreassembly can be carried out in multiple rounds, if desired. Theresulting reassembled genes constitute a library ofbutyrylcholinesterase variants that can be used in the inventioncompositions and methods.

Thus, the invention also provides nucleic acids shown as SEQ ID NOS: SEQID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33,35, 37, 39, 41, and 51, respectively, or fragments thereof, which encodethe butyrylcholinesterase variants encompassing the same orsubstantially the same amino acid sequences shown as SEQ ID NOS: 2, 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42and 52, respectively.

The invention nucleic acids encoding butyrylcholinesterase variants canbe expressed in a variety of eukaryotic cells. For example, the nucleicacids can be expressed in mammalian cells, insect cells, plant cells,and non-yeast fungal cells. Mammalian cell lines useful for expressingthe invention library of nucleic acids encoding butyrylcholinesterasevariants include, for example, Chinese Hamster Ovary (CHO), human T293and Human NIH 3T3 cell lines. Expression of the invention library ofnucleic acids encoding butyrylcholinesterase variants can be achieved byboth stable or transient cell transfection (see Example III, Table 3).

The incorporation of variant nucleic acids or heterologous nucleic acidfragments at an identical site in the genome functions to createisogenic cell lines that differ only in the expression of a particularvariant or heterologous nucleic acid. Incorporation at a single siteminimizes positional effects from integration at multiple sites in agenome that affect transcription of the mRNA encoded by the nucleic acidand complications from the incorporation of multiple copies orexpression of more than one nucleic acid species per cell. Techniquesknown in the art that can be used to target a variant or a heterologousnucleic acid to a specific location in the genome include, for example,homologous recombination, retroviral targeting and recombinase-mediatedtargeting.

One approach for targeting variant or heterologous nucleic acids to asingle site in the genome uses Cre recombinase to target insertion ofexogenous DNA into the eukaryotic genome at a site containing a sitespecific recombination sequence (Sauer and Henderson, Proc. Natl. Acad.Sci. USA, 85:5166-5170 (1988); Fukushige and Sauer, Proc. Natl. Acad.Sci. U.S.A. 89:7905-7909 (1992); Bethke and Sauer, Nuc. Acids Res.,25:2828-2834 (1997)). In addition to Cre recombinase, Flp recombinasecan also be used to target insertion of exogenous DNA into a particularsite in the genome (Dymecki, Proc. Natl. Acad. Sci. U.S.A. 93:6191-6196(1996)). The target site for Flp recombinase consists of 13 base-pairrepeats separated by an 8 base-pair spacer:5′-GAAGTTCCTATTC[TCTAGAAA]GTATAGGAACTTC-3′. As described herein, thebutyrylcholinesterases designated SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14,16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 52, wereobtained by transfection of variant libraries corresponding to theregions set forth in Table 2 of human butyrylcholinesterase intomammalian cells using Flp recombinase and the human 293T cell line. Itis understood that any combination of site-specific recombinase andcorresponding recombination site can be used in methods of the inventionto target a nucleic acid to a particular site in the genome.

A suitable recombinase can be encoded on a vector that is co-transfectedwith a vector containing a nucleic acid encoding a butyrylcholinesterasevariant. Alternatively, the expression element of a recombinase can beincorporated into the same vector expressing a nucleic acid encoding abutyrylcholinesterase variant. In addition to simultaneouslytransfecting the nucleic acid encoding a recombinase with the nucleicacids encoding a butyrylcholinesterase variant, a vector encoding therecombinase can be transfected into a cell, and the cells can beselected for expression of recombinase. A cell stably expressing therecombinase can subsequently be transfected with nucleic acids encodingvariant nucleic acids.

As disclosed herein, the precise site-specific DNA recombinationmediated by Cre recombinase can be used to create stable mammaliantransformants containing a single copy of exogenous DNA encoding abutyrylcholinesterase variant. As exemplified below, the frequency ofCre-mediated targeting events can be enhanced substantially using amodified doublelox strategy. The doublelox strategy is based on theobservation that certain nucleotide changes within the core region ofthe lox site alter the site selection specificity of Cre-mediatedrecombination with little effect on the efficiency of recombination(Hoess et al., Nucleic Acids Res. 14:2287-2300 (1986)). Incorporation ofloxP and an altered loxP site, termed lox511, in both the targetingvector and the host cell genome results in site-specific recombinationby a double crossover event. The doublelox approach increases therecovery of site-specific integrants by 20-fold over the singlecrossover insertional recombination, increasing the absolute frequencyof site-specific recombination such that it exceeds the frequency ofillegitimate recombination (Bethke and Sauer, Nuc. Acids Res.,25:2828-2834 (1997)).

Following the expression of a library of butyrylcholinesterase variantsin a mammalian cell line, randomly selected clones can be sequenced andscreened for increased cocaine hydrolysis activity. Methods forsequencing selected clones are well known to those of skill in the artand are described, for example, in Sambrook et al., supra, 1989, and inAusubel et al., supra, 2000. Selecting a suitable method for measuringthe cocaine hydrolysis activity of a butyrylcholinesterase variantdepends on a variety of factors such as, for example, the amount of thebutyrylcholinesterase variant that is available. The cocaine hydrolysisactivity of a butyrylcholinesterase variant can be measured, forexample, by spectrophotometry, by a microtiter-based assay utilizing apolyclonal anti-butyrylcholinesterase antibody to uniformly capture thebutyrylcholinesterase variants and by high-performance liquidchromatography (HPLC).

Enhanced cocaine hydrolysis activity of a butyrylcholinesterase variantcompared to butyrylcholinesterase can be determined by a comparison ofcatalytic efficiencies. Clones expressing butyrylcholinesterase variantsexhibiting increased cocaine hydrolysis activity can be sequenced toconfirm the precise location and nature of the mutation. To ensure thata library of butyrylcholinesterase variants has been screenedexhaustively, screening of each library can be continued until clonesencoding identical butyrylcholinesterase amino acid alterations havebeen identified on multiple occasions.

Clones expressing a butyrylcholinesterase variant with increased cocainehydrolysis activity can be used to establish larger-scale culturessuitable for purifying larger quantities of the butyrylcholinesterase. Abutyrylcholinesterase variant of interest can be cloned into anexpression vector and used to transfect a cell line, which cansubsequently be expanded. Those skilled in the art will know what typeof expression vector is suitable for a particular application. Abutyrylcholinesterase variant exhibiting increased cocaine hydrolysisactivity can be cloned, for example, into an expression vector carryinga gene that confers resistance to a particular chemical agent to allowpositive selection of the transfected cells. An expression vectorsuitable for transfection of, for example, mammalian cell lines cancontain a promoter such as the cytomegalovirus (CMV) promoter forselection in mammalian cells. As described herein, abutyrylcholinesterase variant can be cloned into a mammalian expressionvector and transfected into Chinese Hamster Ovary cells (CHO).Expression vectors suitable for expressing a butyrylcholinesterasevariant are well known in the art and commercially available.

Clones expressing butyrylcholinesterase variants can be selected andtested for cocaine hydrolysis activity. Cells carrying clones exhibitingenhanced cocaine hydrolysis activity can be expanded by routine cellculture systems to produce larger quantities of a butyrylcholinesterasevariant of interest. The concentrated recombinant butyrylcholinesterasevariant can be harvested and purified by methods well known in the artand described, for example, by Masson et al., Biochemistry 36: 2266-2277(1997), which is incorporated herein by reference.

A butyrylcholinesterase variant exhibiting increased cocaine hydrolysisactivity in vitro can be utilized for the treatment of cocaine toxicityand addiction in vivo. The potency for treating cocaine toxicity of abutyrylcholinesterase variant exhibiting increased cocaine hydrolysisactivity in vitro can be tested using an acute overdose animal model asdisclosed herein (see Example VI). In addition, animal models ofreinforcement and discrimination are used to predict the efficacy of abutyrylcholinesterase variant for treatment of cocaine addiction asdisclosed below (see Example VI). Suitable animal subjects for overdoseas well as reinforcement and discrimination models are known in the artand include, for example, rodent and primate models. Abutyrylcholinesterase variant effective in reducing either cocainetoxicity or cocaine addiction in one or more animal models can be usedto treat a cocaine-induced condition by administering an effectiveamount of the butyrylcholinesterase variant to an individual.

A butyrylcholinesterase variant having an increased serum half-life canbe useful for testing a butyrylcholinesterase variant in a subject ortreating a cocaine-induced condition in an individual. Useful methodsfor increasing the serum half-life of a butyrylcholinesterase variantinclude, for example, conversion of the butyrylcholinesterase variantinto a tetramer, covalently attaching synthetic and natural polymerssuch as polyethylene glycol (PEG) and dextrans to the truncatedbutyrylcholinesterase variant, liposome formulations, or expression ofthe enzyme as an Ig-fusion protein. Furthermore, conversion of abutrylcholinesterase variant into a tetramer can be achieved byco-transfecting the host cell line with the COLQ gene as well as byaddition of poly-L-proline to the media of transfected cells. These andother methods known in the art for increasing the serum half-life of abutyrylcholinesterase variant are useful for testing abutyrylcholinesterase variant in an animal subject or treating acocaine-induced condition in an individual.

The invention also provides a method of hydrolyzing a cocaine-basedbutyrylcholinesterase substrate including contacting abutyrylcholinesterase substrate with a butyrylcholinesterase variantselected from the group shown as SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16,18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42 and 52, underconditions that allow hydrolysis of cocaine into metabolites, whereinthe butyrylcholinesterase variant exhibits increased cocaine hydrolysisactivity compared to butyrylcholinesterase as described herein for eachof these variants.

The invention further provides a method of treating a cocaine-inducedcondition including administering to an individual an effective amountof the butyrylcholinesterase variant selected from the group shown asSEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,34, 36, 38, 40, 42 and 52, wherein the butyrylcholinesterase variantexhibits increased cocaine hydrolysis activity compared tobutyrylcholinesterase as described herein for each of these variants.

As described herein, a butyrylcholinesterase variant exhibitingincreased cocaine hydrolysis activity can hydrolyze a cocaine-basedbutyrylcholinesterase substrate in vitro as well as in vivo. Acocaine-based butyrylcholinesterase substrate can be contacted with abutyrylcholinesterase variant of the invention in vitro, for example, byadding the substrate to supernatant isolated from cultures ofbutyrylcholinesterase variant library clones. Alternatively, thebutyrylcholinesterase variant can be purified prior to being contactedby the substrate. Appropriate medium conditions in which to contact acocaine-based substrate with a butyrylcholinesterase variant of theinvention are readily determined by those skilled in the art. Forexample, 100 μM cocaine in 10 mM Tris at pH 7.4 can be contacted with abutyrylcholinesterase variant at 37° C. As described below,butyrylcholinesterase variants from culture supernatants can further beimmobilized using a capture agent, such as an antibody prior to beingcontacted with a substrate, which allows for removal of culturesupernatant components and enables contacting of the immobilizedvariants with substrate in the absence of contaminants. Followingcontacting of a butyrylcholinesterase variant of the invention with acocaine-based substrate, cocaine hydrolysis activity can be measured bya variety of methods known in the art and described herein, for example,by high-performance liquid chromatography or the isotope tracer cocainehydrolysis assay.

The invention also provides a method of treating cocaine overdose aswell as cocaine addiction in an individual by administering atherapeutically effective amount of the butyrylcholinesterase variant.Treatment of a cocaine-induced condition encompasses prophylacticapplications of the invention method in which the invention variant isadministered to an individual predicted to be exposed to cocaine at afuture time. In prophylactic embodiments of the invention method, atherapeutically effective amount of the butyrylcholinesterase variant isadministered prior to cocaine-exposure. As demonstrated in FIGS. 8 and12 for the A328W/Y332M/S287G/F227A variant (SEQ ID NO: 52),pre-treatment with an invention variant has a therapeutic effect bydecreasing cocaine-toxicity in general as well as by delaying thetime-of-onset of symptoms associated with cocaine-induced toxicity.

The dosage of a butyrylcholinesterase variant required to be effectivedepends, for example, on whether an acute overdose or chronic addictionis being treated, the route and form of administration, the potency andbio-active half-life of the molecule being administered, the weight andcondition of the individual, and previous or concurrent therapies. Theappropriate amount considered to be an effective dose for a particularapplication of the method can be determined by those skilled in the art,using the teachings and guidance provided herein. For example, theamount can be extrapolated from in vitro or in vivobutyrylcholinesterase assays described herein. One skilled in the artwill recognize that the condition of the individual needs to bemonitored throughout the course of treatment and that the amount of thecomposition that is administered can be adjusted accordingly.

For treating cocaine-overdose, a therapeutically effective amount of abutyrylcholinesterase variant of the invention can be, for example,between about 0.1 mg/kg to 0.15 mg/kg body weight, for example, betweenabout 0.15 mg/kg to 0.3 mg/kg, between about 0.3 mg/kg to 0.5 mg/kg orpreferably between about 1 mg/kg to 5 mg/kg, depending on the treatmentregimen. For example, if a butyrylcholinesterase variant is administeredto an individual symptomatic of cocaine overdose a higher one-time doseis appropriate, while an individual symptomatic of chronic cocaineaddiction may be administered lower doses from one to several times aday, weekly, monthly or less frequently. Similarly, formulations thatallow for timed-release of a butyrylcholinesterase variant would providefor the continuous release of a smaller amount of abutyrylcholinesterase variant to an individual treated for chroniccocaine addiction. It is understood, that the dosage of abutyrylcholinesterase variant has to be adjusted based on the catalyticactivity of the variant, such that a lower dose of a variant exhibitingsignificantly enhanced cocaine hydrolysis activity can be administeredcompared to the dosage necessary for a variant with lower cocainehydrolysis activity.

The time for commencing treatment with a butyrylcholinesterase variantcan be prior to contact with the cocaine-based substrate, for example,cocaine, or can be following contact with the cocaine-based substrate.For treatment of a cocaine overdose it is desirable to administer theinvention variant as soon as possible after contact so as to maximizetherapeutic effect. As shown in FIG. 9 for the butyrylcholinesterasevariant designated A328W/Y332M/S287G/F227A (SEQ ID NO: 52), the effectof therapeutic treatment on cocaine-induced toxicity maintains fullprotection when administered at 8 minutes after contact and decreasedwhen administered at later time points as a result of onset ofphysiologically irreversible symptoms of cocaine-toxicity. Nevertheless,as shown in FIG. 9, treatment with an invention variant is effectiveeven after onset of symptoms associated with cocaine-induced toxicity.Therefore, for treatment of cocaine-induced toxicity abutyrylcholinesterase variant of the invention can be administered priorto contact as well as following contact, for example, within seconds orminutes, including after about 1 minute or less, about 2 minutes orless, about 3 minutes or less, about 4 minutes or less, about 5 minutesor less, about 6 minutes or less, about 7 minutes or less, about 8minutes or less, about 9 minutes or less, about 10 minutes or less,about 11 minutes or less, about 12 minutes or less, about 13 minutes orless, about 14 minutes or less, about 15 minutes or less, about 16minutes or less, about 17 minutes or less, about 18 minutes or less,about 19 minutes or less, about 20 minutes or less, about 21 minutes orless, about 22 minutes or less, about 23 minutes or less, about 24minutes or less, about 25 minutes or less, about 30 minutes or less,about 35 minutes or less, about 40 minutes or less, about 45 minutes orless, about 50 minutes or less, about 55 minutes or less, about 60minutes or less, about 90 minutes or less, and about 120 minutes orless.

It is understood that the timing of effective treatment for toxicity islimited only by the irreversibility of the physiological symptoms anddamage associated with the cocaine-induced toxicity. As shown in FIG. 9,effective treatment was fully sustained through onset of particularsymptoms, in particular, the first set of slight convulsions, and wassignificantly sustained even through the onset of more advancedsymptoms, in particular, the second set of convulsions. Thus, it isfurther understood that effectiveness of treatment can be generallyassociated with time after contact, but also can be viewed as itcorresponds to the progression of symptoms. For example, it can beuseful to observe progression of symptoms following contact to chooseparticular treatment parameters based on observations regarding theprogression of symptoms rather than on progression of time.

A butyrylcholinesterase variant can be delivered systemically, such asintravenously or intraarterially. A butyrylcholinesterase variant can beprovided in the form of isolated and substantially purified polypeptidesand polypeptide fragments in pharmaceutically acceptable formulationsusing formulation methods known to those of ordinary skill in the art.These formulations can be administered by standard routes, including forexample, topical, transdermal, intraperitoneal, intracranial,intracerebroventricular, intracerebral, intravaginal, intrauterine,oral, rectal or parenteral (e.g., intravenous, intraspinal, subcutaneousor intramuscular) routes. In addition, a butyrylcholinesterase variantcan be incorporated into biodegradable polymers allowing for sustainedrelease of the compound useful for treating individual symptomatic ofcocaine addiction. Biodegradable polymers and their use are described,for example, in detail in Brem et al., J. Neurosurg. 74:441-446 (1991),which is incorporated herein by reference.

A butyrylcholinesterase variant can be administered as a solution orsuspension together with a pharmaceutically acceptable medium. Such apharmaceutically acceptable medium can be, for example, water, sodiumphosphate buffer, phosphate buffered saline, normal saline or Ringer'ssolution or other physiologically buffered saline, or other solvent orvehicle such as a glycol, glycerol, an oil such as olive oil or aninjectable organic ester. A pharmaceutically acceptable medium canadditionally contain physiologically acceptable compounds that act, forexample, to stabilize or increase the absorption of thebutyrylcholinesterase variant. Such physiologically acceptable compoundsinclude, for example, carbohydrates such as glucose, sucrose ordextrans; antioxidants such as ascorbic acid or glutathione; chelatingagents such as EDTA, which disrupts microbial membranes; divalent metalions such as calcium or magnesium; low molecular weight proteins; lipidsor liposomes; or other stabilizers or excipients.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions such as the pharmaceuticallyacceptable mediums described above. The solutions can additionallycontain, for example, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient. Otherformulations include, for example, aqueous and non-aqueous sterilesuspensions which can include suspending agents and thickening agents.The formulations can be presented in unit-dose or multi-dose containers,for example, sealed ampules and vials, and can be stored in alyophilized condition requiring, for example, the addition of thesterile liquid carrier, immediately prior to use. Extemporaneousinjection solutions and suspensions can be prepared from sterilepowders, granules and tablets of the kind previously described.

The butyrylcholinesterase variant of the invention can further beutilized in combination therapies with other therapeutic agents.Combination therapies that include a butyrylcholinesterase variant canconsist of formulations containing the variant and the additionaltherapeutic agent individually in a suitable formulation. Alternatively,combination therapies can consist of fusion proteins, where thebutyrylcholinesterase variant is linked to a heterologous protein, suchas a therapeutic protein.

The butyrylcholinesterase variant of the invention also can be deliveredto an individual by administering an encoding nucleic acid for thepeptide or variant. The encoding nucleic acids for thebutyrylcholinesterase variant of the invention are useful in conjunctionwith a wide variety of gene therapy methods known in the art fordelivering a therapeutically effective amount of the polypeptide orvariant. Using the teachings and guidance provided herein, encodingnucleic acids for a butyrylcholinesterase variant can be incorporatedinto a vector or delivery system known in the art and used for deliveryand expression of the encoding sequence to achieve a therapeuticallyeffective amount. Applicable vector and delivery systems known in theart include, for example, retroviral vectors, adenovirus vectors,adenoassociated virus, ligand conjugated particles and nucleic acids fortargeting, isolated DNA and RNA, liposomes, polylysine, and celltherapy, including hepatic cell therapy, employing the transplantationof cells modified to express a butyrylcholinesterase variant, as well asvarious other gene delivery methods and modifications known to thoseskilled in the art, such as those described in Shea et al., NatureBiotechnology 17:551-554 (1999), which is incorporated herein byreference.

Specific examples of methods for the delivery of a butyrylcholinesterasevariant by expressing the encoding nucleic acid sequence are well knownin art and described in, for example, U.S. Pat. No. 5,399,346; U.S. Pat.Nos. 5,580,859; 5,589,466; 5,460,959; 5,656,465; 5,643,578; 5,620,896;5,460,959; 5,506,125; European Patent Application No. EP 0 779 365 A2;PCT No. WO 97/10343; PCT No. WO 97/09441; PCT No. WO 97/10343, all ofwhich are incorporated herein by reference. Other methods known to thoseskilled in the art also exist and are similarly applicable for thedelivery of a butyrylcholinesterase variant by expressing the encodingnucleic acid sequence.

In addition to the treatment of cocaine-induced conditions such ascocaine overdose or cocaine addiction, a butyrylcholinesterase can alsobe administered prophylactically to avoid the onset of a cocaineoverdose upon subsequent entry of cocaine into the bloodstream. It isfurther contemplated that a butyrylcholinesterase variant exhibitingincreased cocaine hydrolysis activity of the invention can havediagnostic value by providing a tool for efficiently determining thepresence and amount of a cocaine-induced substance in a medium.

It is understood that modifications that do not substantially affect theactivity of the various embodiments of this invention are also includedwithin the definition of the invention provided herein. Accordingly, thefollowing examples are intended to illustrate but not limit the presentinvention.

EXAMPLE I Development of a Cocaine Hydrolysis Assay

This example describes the development of a cocaine hydrolysis assaythat permits the efficient analysis of hundreds of butyrylcholinesterasevariants simultaneously.

Development of an Isotope Tracer Cocaine Hydrolysis Assay.

For the purpose of validating new cocaine hydrolysis assays,butyrylcholinesterase hydrolysis of cocaine was first measured asdescribed previously (Xie et al., Mol. Pharmacol. 55:83-91 (1999)),using high-performance liquid chromatography (HPLC). Briefly, reactionscontaining 100 μM cocaine in 10 mM Tris, pH 7.4 were initiated by theaddition of horse butyrylcholinesterase (ICN Pharmaceuticals, Inc.,Costa Mesa, Calif.) and incubated 2-4 hours at 37° C. Following theincubation, the pH was adjusted to 3, and the sample was filtered.Subsequently, the sample was applied to a Hypersil ODS-C 18 reversedphase column (Hewlett Packard, Wilmington, Del.) previously equilibratedwith an 80:20 mixture of 0.05 M potassium phosphate, pH 3.0 andacetonitrile. The isocratic elution of cocaine, benzoylecgonine, andbenzoic acid was quantitated at 220 nm. Measurement of the formation ofecgonine methyl ester and benzoic acid was dependent both on the amountof butyrylcholinesterase in the reaction and on the time of reaction.

At the conclusion of the isotope tracer assay, an aliquot of thereaction mix is acidified in order to take advantage of the solubilitydifference between the product and the substrate at pH 3.0. At pH 3.0,[3H]-benzoic acid (pKa=4.2) is soluble in a scintillation cocktailconsisting of 2,5-diphenyloxazole (PPO) and[1,4-bis-2-(4-methyl-8-phenyloxazolyl)-benzene] (POPOP)(PPO-dimethyl-POPOP scintillation fluor, Research Products InternationalCorp., Mt. Prospect, Ill.) while [3H]-cocaine is not. The signalgenerated by acidified reaction mixture from enzyme blanks was less than2% of the total dpm placed in the fluor, consistent with cocaine beinginsoluble in PPO-dimethyl-POPOP.

The isotope tracer cocaine hydrolysis assay was validated by directcomparison with the established HPLC assay and the accuracy of theisotope assay was demonstrated by determining the K_(m) value for horsebutyrylcholinesterase. The rate of cocaine hydrolysis, determined bymeasuring the rate of formation of benzoic acid was quantitated both byHPLC and the isotope tracer assay in reactions containing variableamounts of butyrylcholinesterase. Formation of [³H]-benzoic acid wasdependent on the length of assay incubation and on the amount ofbutyrylcholinesterase added. Good correlation between the establishedHPLC assay and the isotope tracer assay was observed, as demonstrated byplotting the quantitation of benzoic acid formation measured by HPLCversus the benzoic acid formation measured in the isotope assay (seeFIG. 5A; r²=0.979). To demonstrate the precision and sensitivity of theisotope assay the amount of cocaine was varied and the K_(m) wasdetermined using the Lineweaver-Burk double-reciprocal plot of cocainehydrolysis by horse butyrylcholinesterase depicted in FIG. 5B. Velocitywas calculated as cpm benzoic acid formed×10⁻⁵ following a 2 hourincubation at 37° C. Based on these data the K_(m) for cocainehydrolysis is approximately 37.6 μM (×intercept=−l/k_(m)), which is inclose agreement with previously published values of 38 μM (Gatley,supra, 1991) and 45±5 μM (Xie et al., supra, 1999) for horsebutyrylcholinesterase.

Immobilization of Active Butyrylcholinesterase

The supernatants isolated from each of the butyrylcholinesterase variantlibrary clones contains variable butyrylcholinesterase enzymeconcentrations. Consequently, the cocaine hydrolysis activity measuredfrom equal volumes of culture supernatants from distinctbutyrylcholinesterase variant clones reflects the expression level aswell as the enzyme activity. In order to be able to compare equal enzymeconcentrations and more rapidly identify variants with the desiredactivity, butyrylcholinesterase from culture supernatants areimmobilized using a capture reagent, such as an antibody, that issaturated at low butyrylcholinesterase concentrations as describedpreviously by Watkins et al., Anal. Biochem. 253: 37-45 (1997). As aresult, butyrylcholinesterase from dilute samples is concentrated anduniform quantities of different butyrylcholinesterase variant clones areimmobilized, regardless of the initial concentration ofbutyrylcholinesterase in the culture supernatant. Subsequently, unboundbutyrylcholinesterase and other culture supernatant components thatpotentially interfere with the assay (such as unrelated serum orcell-derived proteins with significant esterase activity) are washedaway and the activity of the immobilized butyrylcholinesterase isdetermined by measuring the formation of benzoic acid as describedabove.

To assess the efficiency of the above assay, efficient capture of humanbutyrylcholinesterase, as well as a truncated soluble monomeric form ofhuman butyrylcholinesterase (Blong et al., Biochem. J. 327: 747-757(1997)), was demonstrated in a microtiter format using a commerciallyavailable rabbit anti-human cholinesterase polyclonal antibody (DAKO,Carpinteria, Calif.)(FIG. 6). In order to determine the optimalconditions for capturing butyrylcholinesterase a microtiter plate wascoated with increasing quantities of rabbit anti-butyrylcholinesterase,was blocked, and incubated with varying amounts of culture supernatant.The amount of active butyrylcholinesterase captured was determinedcalorimetrically using an assay that measures butyrylthiocholinehydrolysis at 405 nm in the presence of dithiobisnitrobenzoic acid (Xieet al., supra, 1999). Subsequently, the butyrylcholinesterase activitycaptured from dilutions of culture supernatants from cells expressingeither the wild-type human butyrylcholinesterase or the monomerictruncated version was measured. The rabbit anti-butyrylcholinesterasecapture antibody was saturated by the butyrylcholinesterase present in25 μl of culture supernatant with greater butyrylcholinesterase activitybeing captured from supernatant containing the full length wild-typeform of the enzyme (FIG. 6, compare filled circles with open circles).Unbound material was removed by washing with 100 mM Tris, pH 7.4 and theamount of active butyrylcholinesterase captured was quantitated bymeasuring butyrylthiocholine hydrolysis. Butyrylcholinesterase isexpressed in culture supernatants at quantities sufficient to saturate apolyclonal anti-butyrylcholinesterase antibody on a microtiter plate. Inaddition, the captured enzyme is active, as demonstrated by thehydrolysis of butyrylthiocholine.

Measurement of Cocaine Hydrolysis with Isotope Tracer Assay andImmobilized Butyrylcholinesterase

The optimal conditions for immobilization of activebutyrylcholinesterase are used in conjunction with the cocaine isotopetracer assay to measure the cocaine hydrolysis activity in a microtiterformat. The assay is characterized by determining the K_(m) for cocainehydrolysis activity, as described above. At least three approaches areused to either increase the assay sensitivity or the assay signal.

First, longer assay incubation times that proportionately increase thesignal can be used. Second, the sensitivity of the assay can be enhancedby increasing the specific activity of the radiolabeled cocainesubstrate. Third, a previously identified butyrylcholinesterase mutantwhich is 4-fold more efficient for cocaine hydrolysis can used (Xie etal., supra, 1999), which in conjunction with doubling the assayincubation time and increasing the specific activity of the cocaine10-fold, can increase the assay signal about 80-fold.

EXAMPLE II Synthesis and Characterization of ButyrylcholinesteraseVariant Libraries

This example describes the synthesis and characterization ofbutyrylcholinesterase variant libraries expressed in mammalian cells.

In order to facilitate the synthesis of libraries ofbutyrylcholinesterase variants, DNA encoding wild-type humanbutyrylcholinesterase, a truncated, enzymatically active, monomericversion of human butyrylcholinesterase, and the A328Y mutant thatdisplays a four-fold increased cocaine hydrolysis activity are clonedinto a modified doublelox targeting vector, using unique restrictionsites. In preliminary assays the wild-type human butyrylcholinesterasewas captured more efficiently and, therefore, serves as the initial DNAtemplate for the synthesis of libraries of butyrylcholinesterasevariants.

Synthesis of Focused Libraries of Butyrylcholinesterase Variants byCodon-Based Mutagenesis

A variety of information can be used to focus the synthesis of theinitial libraries of butyrylcholinesterase variants to discreet regions.For example, butyrylcholinesterase and Torpedo acetylcholinesterase(AChE) share a high degree of homology (53% identity). Furthermore,residues 4 to 534 of Torpedo AChE can be aligned with residues 2 to 532of butyrylcholinesterase without deletions or insertions. The catalytictriad residues (butyrylcholinesterase residues Ser198, Glu325, andHis438) and the intrachain disulfides are all in the same positions. Dueto the high degree of similarity between these proteins, a refined 2.8—Åx-ray structure of Torpedo AChE (Sussman et al., Science 253: 872-879(1991)) has been used to model butyrylcholinesterase structure (Harel etal., supra, 1992)).

Studies with cholinesterases have revealed that the catalytic triad andother residues involved in ligand binding are positioned within a deep,narrow, active-site gorge rich in hydrophobic residues (reviewed inSoreq et al., Trends Biochem. Sci. 17:353-358 (1992)). The sites ofseven focused libraries of butyrylcholinesterase variants (FIG. 2,underlined residues) were selected to include amino acids determined tobe lining the active site gorge (FIG. 2, hydrophobic active site gorgeresidues are shaded).

In addition to the structural modeling of butyrylcholinesterase,butyrylcholinesterase biochemical data was integrated into the librarydesign process. For example, characterization of naturally occurringbutyrylcholinesterases with altered cocaine hydrolysis activity andsite-directed mutagenesis studies provide information regarding aminoacid positions and segments important for cocaine hydrolysis activity(reviewed in Schwartz et al., Pharmac. Ther. 67: 283-322(1995)).Moreover, comparison of sequence and cocaine hydrolysis data ofbutyrylcholinesterases from different species can also provideinformation regarding regions important for cocaine hydrolysis activityof the molecule based on comparison of the cocaine hydrolysis activitiesof these butyrylcholinesterases. The previously identified A328Y mutantis present in the library corresponding to region 6 and serves as acontrol to demonstrate the quality of the library synthesis andexpression in mammalian cells as well as the sensitivity of themicrotiter-based cocaine hydrolysis assay.

TABLE 2 Butyrylcholinesterase Regions Predicted to be Important forCatalytic Efficiency. Region Location Length 1 68-82 15 2 110-121 12 3194-201 8 4 224-234 11 5 277-289 13 6 327-332 6 7 429-442 14

The seven regions of butyrylcholinesterase selected for focused librarysynthesis span residues that include the 8 aromatic active site gorgeresidues (W82, W112, Y128, W231, F329, Y332, W430 and Y440) as well astwo of the catalytic triad residues. The integrity of intrachaindisulfide bonds, located between ⁶⁵Cys-⁹²Cys, ²⁵²Cys⁻²⁶³Cys, and⁴⁰⁰Cys⁻⁵¹⁹Cys is maintained to ensure functional butyrylcholinesterasestructure. In addition, putative glycosylation sites (N-X-S/T) locatedat residues 17, 57, 106, 241, 256, 341, 455, 481, 485, and 486 also areavoided in the library synthesis. In total, the seven focused librariesspan 79 residues, representing approximately 14% of thebutyrylcholinesterase linear sequence, and result in the expression ofabout 1500 distinct butyrylcholinesterase variants.

Libraries of nucleic acids corresponding to the seven regions of humanbutyrylcholinesterase to be mutated are synthesized by codon-basedmutagenesis, as described above and as depicted schematically in FIG. 7.Briefly, multiple DNA synthesis columns are used for synthesizing theoligonucleotides by β-cyanoethyl phosphoramidite chemistry, as describedpreviously by Glaser et al., supra, 1992. In the first step,trinucleotides encoding for the amino acids of butyrylcholinesterase aresynthesized on one column while a second column is used to synthesizethe trinucleotide NN(G/T), where N is a mixture of dA, dG, dC, and, dTcyanoethyl phosphoramidities. Using the trinucleotide NN(G/T) results inthorough mutagenesis with minimal degeneracy, accomplished through thesystematic expression of all twenty amino acids at every position.

Following the synthesis of the first codon, resins from the two columnsare mixed together, divided, and replaced in four columns. By addingadditional synthesis columns for each codon and mixing the column resinsin the manner illustrated in FIG. 7, pools of degenerateoligonucleotides will be segregated based on the extent of mutagenesis.The resin mixing aspect of codon-based mutagenesis makes the processrapid and cost-effective because it eliminates the need to synthesizemultiple oligonucleotides. In the present study, the pool ofoligonucleotides encoding single amino acid mutations is used tosynthesize focused butyrylcholinesterase libraries.

The oligonucleotides encoding the butyrylcholinesterase variantscontaining a single amino acid mutation is cloned into the doubleloxtargeting vector using oligonucleotide-directed mutagenesis (Kunkel,supra, 1985). To improve the mutagenesis efficiency and diminish thenumber of clones expressing wild-type butyrylcholinesterase, thelibraries are synthesized in a two-step process. In the first step, thebutyrylcholinesterase DNA sequence corresponding to each library site isdeleted by hybridization mutagenesis. In the second step,uracil-containing single-stranded DNA for each deletion mutant, onedeletion mutant corresponding to each library, is isolated and used astemplate for synthesis of the libraries by oligonucleotide-directedmutagenesis. This approach has been used routinely for the synthesis ofantibody libraries and results in more uniform mutagenesis by removingannealing biases that potentially arise from the differing DNA sequenceof the mutagenic oligonucleotides. In addition, the two-step processdecreases the frequency of wild-type sequences relative to the variantsin the libraries, and consequently makes library screening moreefficient by eliminating repetitious screening of clones encodingwild-type butyrylcholinesterase.

The quality of the libraries and the efficiency of mutagenesis ischaracterized by obtaining DNA sequence from approximately 20 randomlyselected clones from each library. The DNA sequences demonstrate thatmutagenesis occurs at multiple positions within each library and thatmultiple amino acids were expressed at each position. Furthermore, DNAsequence of randomly selected clones demonstrates that the librariescontain diverse clones and are not dominated by a few clones.

Optimization of Transfection Parameters for Site-Specific Integration

Optimization of transfection parameters for Cre-mediated site-specificintegration was achieved utilizing Bleomycin Resistance Protein (BRP)DNA as a model system.

Cre recombinase is a well-characterized 38-kDa DNA recombinase (Abremskiet al., Cell 32:1301-1311 (1983)) that is both necessary and sufficientfor sequence-specific recombination in bacteriophage P1. Recombinationoccurs between two 34-base pair loxP sequences each consisting of twoinverted 13-base pair recombinase recognition sequences that surround acore region (Sternberg and Hamilton, J. Mol. Biol. 150:467-486 (1981a);Sternberg and Hamilton, J. Mol. Biol., 150:487-507 (1981b)). DNAcleavage and strand exchange occurs on the top or bottom strand at theedges of the core region. Cre recombinase also catalyzes site-specificrecombination in eukaryotes, including both yeast (Sauer, Mol. Cell.Biol. 7:2087-2096 (1987)) and mammalian cells (Sauer and Henderson,Proc. Natl. Acad. Sci. USA, 85:5166-5170 (1988); Fukushige and Sauer,Proc. Natl. Acad. Sci. U.S.A. 89:7905-7909 (1992); Bethke and Sauer,Nuc. Acids Res., 25:2828-2834 (1997)).

Calcium phosphate transfection of 13-1 cells was previously demonstratedto result in targeted integration in 1% of the viable cells plated(Bethke and Sauer, Nuc. Acids Res., 25:2828-2834 (1997)). Therefore,initial studies were conducted using calcium phosphate to transfect 13-1cells with 4 μg pBS185 and 10, 20, 30, or 40 μg of pBS397-fl(+)/BRP. Thetotal level of DNA per transfection was held constant using unrelatedpBluescript II KS DNA (Stratagene; La Jolla, Calif.), and transformantswere selected 48 hours later by replating in media containing 400 μg/mlgeneticin. Colonies were counted 10 days later to determine theefficiency of targeted integration. Optimal targeted integration wastypically observed using 30 μg of targeting vector and 4 μg of Crerecombinase vector pBS185, consistent with the 20 μg targeting vectorand 5 μg of pBS185 previously reported (Bethke and Sauer, Nuc. AcidsRes., 25:2828-2834 (1997)). The frequency of targeted integrationobserved was generally less than 1%. Despite the sensitivity of thecalcium phosphate methodology to the amount of DNA used and the bufferpH, targeted integration efficiencies observed were sufficient toexpress the protein libraries.

As shown in Table 3, several cell lines as well as other transfectionmethods were also characterized. As disclosed herein, Flp recombinasealso can be used to target insertion of exogenous DNA into a particularsite in the genome as described by Dymecki, supra, 1996. The target sitefor Flp recombinase consists of 13 base-pair repeats separated by an 8base-pair spacer: 5′-GAAGTTCCTATTC[TCTAGAAA]GTATAGGAACTTC-3′. Briefly,variant libraries corresponding to the region of butyrylcholinesterasecorresponding to amino acids 327 to 332 of butyrylcholinesterase (shownas region 6 in Table 2) were transfected into mammalian cells using flprecombinase and the 293T cell line. The butyrylcholinesterase variantsdesignated SEQ ID NOS: 2, 4, 6 and 8 were identified and characterizedusing the methods described herein utilizing Flp recombinase and the293T human cell line.

In general, lipid-mediated transfection methods are more efficient thanmethods that alter the chemical environment, such as calcium phosphateand DEAE-dextran transfection. In addition, lipid-mediated transfectionsare less affected by contaminants in the DNA preparations, saltconcentration, and pH and thus generally provide more reproducibleresults (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417(1987)). Consequently, a formulation of the neutral lipid dioleoylphosphatidylethanolamine and a cationic lipid, termed GenePORTERtransfection reagent (Gene Therapy Systems; San Diego, Calif.), wasevaluated as an alternative transfection approach. Briefly,endotoxin-free DNA was prepared for both the targeting vectorpBS397-fl(+)/BRP and the Cre recombinase vector pBS185 using theEndoFree Plasmid Maxi kit (QIAGEN; Valencia, Calif.). Next, 5 μg pBS185and varying amounts of pBS397-fl(+)/BRP were diluted in serum-freemedium and mixed with the GenePORTER transfection reagent. The DNA/lipidmixture was then added to a 60-70% confluent monolayer of 13-1 cellsconsisting of approximately 5×10⁵ cells/100-mm dish and incubated at 37°C. Five hours later, fetal calf serum was added to 10%, and the next daythe transfection media was removed and replaced with fresh media.

Transfection of the cells with variable quantities of the targetingvector yielded targeted integration efficiencies ranging from 0.1% to1.0%, with the optimal targeted integration efficiency observed using 5μg each of the targeting vector and the Cre recombinase vector.Lipid-based transfection of the 13-1 host cells under the optimizedconditions resulted in 0.5% targeted integration efficiency beingconsistently observed. A 0.5% targeted integration is slightly less thanthe previously reported 1.0% efficiency (Bethke and Sauer, Nuc. AcidsRes., 25:2828-2834 (1997)), and is sufficient to express large proteinlibraries and allows expressing libraries of protein variants inmammalian cells.

TABLE 3 Expression of a single butyrylcholinesterase variant per cellusing either stable or transient cell transfection. Cell IntegrationIntegration? Integration? Line Expression Method (PCR) (Activity) NIH3T3Transient N/A N/A Transient, (13-1) (lipid-based) very low activityNIH3T3 Stable Cre Yes No measurable (13-1) recombinase activity CHOTransient N/A N/A Transient, (lipid-based) measurable activity(colorimetric and cocaine hydrolysis) 293 Transient N/A N/A Transient,(lipid-based) measurable activity (colorimetric and cocaine hydrolysis)293 Stable Flp Yes Measurable recombinase activity (colorimetric andcocaine hydrolysis)

These results demonstrate optimization of transfection conditions fortargeted insertion in NlH3T3 13-1 cells. Conditions for a simple,lipid-based transfection method that required a small amount of DNA andgenerated reproducible 0.5% targeting efficiency were established.

Expression of Butyrylcholinesterase Variant Libraries in Mammalian Cells

Each of the seven libraries of butyrylcholinesterase variants aretransformed into a host mammalian cell line using the doubleloxtargeting vector and the optimized transfection conditions describedabove. Following Cre-mediated transformation the host cells are platedat limiting dilutions to isolate distinct clones in a 96-well format.Cells with the butyrylcholinesterase variants integrated in the Cre/loxtargeting site are selected with geneticin. Subsequently, the DNAencoding butyrylcholinesterase variants from 20-30 randomly selectedclones from each library are sequenced and analyzed as described above.Briefly, total cellular DNA is isolated from about 10⁴ cells of eachclone of interest using DNeasy Tissue Kits (Qiagen, Valencia, Calif.).Next, the butyrylcholinesterase gene is amplified using PfuTurbo DNApolymerase (Stratagene; La Jolla, Calif.) and an aliquot of the PCRproduct is then used for sequencing the DNA encodingbutyrylcholinesterase variants from randomly selected clones by thefluorescent dideoxynucleotide termination method (Perkin-Elmer, Norwalk,Conn.) using a nested oligonucleotide primer.

As described previously, the sequencing demonstrates uniformintroduction of the library and the diversity of mammalian transformantsresembles the diversity of the library in the doublelox targeting vectorfollowing transformation of bacteria.

TABLE 4 Relative Activity of butyrylcholinesterase variants (WT = 1)with enhanced cocaine hydrolase activity and corresponding codonchanges. Wild-type 1 A199S GCA to TCA 2.5 F227A TTT to GCG 4.1 F227G TTTto GGG 4.0 F227S TTT to AGT 2.3 F227P TTT to CCG 2.9 F227T TTT to ACT1.9 F227C TTT to TGT 1.9 F227M TTT to ATG 1.4 P285Q CCT to CAG 2.4 P285SCCT to AGC 1.9 S287G TCA to GGT 4.1 A328W GCT to TGG 7 V331L GTC to TTGn.d Y332S TAT to TCG n.d Y332M TAT to ATG n.d Y332P TAT to CCA n.dA328W/Y332M/S287G/F227A/A199S 100 A328W/S287G/F227A/A199S 100A328W/S287G/A199S 97 A328W/S287G/F227A 91 A328W/F227A 68 A328W/Y332M 24A328W/Y332P 10 A328W/V331L 16 A328W/Y332S 8

As described herein, a library corresponding to region five ofbutyrylcholinesterase was expressed and individual variants werescreened by measuring the hydrolysis of [³H]-cocaine using themicrotiter assay. The catalytic efficiency (V_(max)/K_(m)) of variantswith enhanced activity were characterized using the microtiter assay todetermine their relative K_(m) and V_(max). Twenty-onebutyrylcholinesterase variants were identified that have enhancedcocaine hydrolase activity: A328W/Y332M(SEQ ID NO: 2), A328W/Y332P (SEQID NO: 4), A328W/V331L (SEQ ID NO: 6) and A328W/Y332S(SEQ ID NO: 8),A328W/Y332M/S287G/F227A/A199S (SEQ ID NO: 10), A328W/S287G/F227A/A199S(SEQ ID NO: 12), A328W/S287G/A199S (SEQ ID NO: 14), A328W/S287G/F227A(SEQ ID NO: 16), A328W/F227A (SEQ ID NO: 18), Y322S (SEQ ID NO: 20),Y332M (SEQ ID NO: 22), Y332P (SEQ ID NO: 24), V331L (SEQ ID NO: 26),F227A (SEQ ID NO: 28), F227G (SEQ ID NO: 30), F227S (SEQ ID NO: 32),F227P (SEQ ID NO: 34), F227T (SEQ ID NO: 36), F227C (SEQ ID NO: 38),F227M (SEQ ID NO: 40), A199S (SEQ ID NO: 42).

EXAMPLE III Characterization of Butyrylcholinesterase Variants thatDisplay Enhanced Cocaine Hydrolysis Activity

This example describes the molecular characterization ofbutyrylcholinesterase variants that display enhanced cocaine hydrolysisactivity in the microtiter assay described below. The cocaine hydrolysisactivity measured in the microtiter assay format is further confirmedusing greater amounts of the butyrylcholinesterase variants of interest.In addition to the microtiter-based assay, the activity of the clones isdemonstrated in solution phase with product formation measured by theHPLC assay to verify the increased cocaine hydrolysis activity of thebutyrylcholinesterase variants and confirm that the enhanced hydrolysisis at the benzoyl ester group.

The kinetic constants for wild-type butyrylcholinesterase and the bestvariants are determined and used to compare the catalytic efficiency ofthe variants relative to wild-type butyrylcholinesterase. K_(m) valuesfor (−)-cocaine are determined at 37° C. V_(max) and K_(m) values arecalculated using Sigma Plot (Jandel Scientific, San Rafael, Calif.). Thenumber of active sites of butyrylcholinesterase is determined by themethod of residual activity using echothiopate iodide or diisopropylfluorophosphates as titrants, as described previously by Masson et al.,Biochemistry 36: 2266-2277 (1997). Alternatively, the number ofbutyrylcholinesterase active sites is estimated using an ELISA toquantitate the mass of butyrylcholinesterase or butyrylcholinesterasevariants present in culture supernatants. Purified humanbutyrylcholinesterase is used as the standard for the ELISA quantitationassay. The catalytic rate constant, k_(cat), is calculated by dividingV_(max) by the concentration of active sites. Finally, the catalyticefficiencies of the best variants are compared to wild-typebutyrylcholinesterase by determining k_(cat)/K_(m) for eachbutyrylcholinesterase variant.

In order to better characterize all the clones expressingbutyrylcholinesterase variants with increased cocaine hydrolysisactivity, the DNA encoding the variants is sequenced. DNA sequencingreveals the precise location and nature of the mutations and thus,quantifies the total number of distinct butyrylcholinesterase variantsidentified. Screening of each library is complete when clones encodingidentical butyrylcholinesterase mutations are identified on multipleoccasions, indicating that the libraries have been screenedexhaustively.

EXAMPLE IV Synthesis and Characterization of CombinatorialButyrylcholinesterase Variant Libraries

This example demonstrates synthesis and characterization ofcombinatorial libraries of butyrylcholinesterase variants expressed inmammalian cells.

The beneficial mutations identified from screening libraries ofbutyrylcholinesterase variants containing a single amino acid mutationare combined in vitro to further improve the butyrylcholinesterasecocaine hydrolysis activity. The positive combination of beneficialmutations designated biochemical additivity has been observed onmultiple occasions. For example, the iterative process of increasingantibody affinity in a stepwise fashion through the accumulation andsubsequent combination of beneficial mutations has led to theidentification of antibodies displaying 500-fold enhanced affinity usingvariant libraries containing less than 2,500 distinct variants.Importantly, the principle of biochemical additivity is not restrictedto improving the affinity of antibodies, and has been exploited toachieve improvements in other physical properties, such asthermostability, catalytic efficiency, or enhanced resistance topesticides.

The best mutations identified from screening the seven focusedbutyrylcholinesterase libraries are used to synthesize a combinatoriallibrary. The number of distinct variants in the combinatorial library isexpected to be small, typically a fraction of the number of distinctvariants from the initial libraries. For example, combinatorial analysisof single mutations at eight distinct sites would require a library thatcontains 2⁸, or 256, unique variants. The combinatorial library issynthesized by oligonucleotide-directed mutagenesis, characterized, andexpressed in the mammalian host cell line. Variants are screened andcharacterized as described above. DNA sequencing reveals additivemutations.

EXAMPLE V Expression and Purification of Butyrylcholinesterase Variants

This example demonstrates the expression in a mammalian cell line andsubsequent purification of butyrylcholinesterase variants.

Clones expressing the most catalytically active butyrylcholinesterasevariants, as well as wild-type butyrylcholinesterase, are used toestablish larger-scale cultures in order to purify quantities of theenzyme necessary for in vivo studies. It is estimated that approximately100 mg each of wild-type butyrylcholinesterase and the optimal variantis required to complete the in vivo toxicity and addiction studies inrats as described below.

The butyrylcholinesterase variants of interest are cloned into thepCMV/Zeo vector (Invitrogen, Carlsbad, Calif.) using unique restrictionsites. The cloning of the variants is verified using restriction mappingand DNA sequencing. Subsequently, the variants are expressed intransfected Chinese Hamster ovary cells CHO Kl(ATCC CCL 61). CHO cellswere selected for expression because butyrylcholinesterase is aglycoprotein and these cells have been previously used for theexpression of recombinant human therapeutic glycoproteins (Goochee etal., Biotechnology 9:1347-1355 (1991); Jenkins and Curling, EnzymeMicrob. Technol. 16:354-364 (1994)) as well as fully active recombinantbutyrylcholinesterase (Masson et al., supra, 1997). Initially, the CHOcells are transiently transfected with all the butyrylcholinesterasevariants to confirm expression of functional butyrylcholinesterase.Subsequently, the cells are stably transfected and clones expressingbutyrylcholinesterase variants are selected using the antibiotic Zeocin(Invitrogen. Carlsbad, Calif.). Colonies are picked with a sterilecotton-tipped stick and transferred to 24-well plates. Thebutyrylcholinesterase expression is measured and the colonies with thehighest activity are further expanded. The kinetic constants of thebutyrylcholinesterase variants are determined to ensure that expressionin CHO cells does not diminish the enzymatic activity compared tobutyrylcholinesterase variants expressed in NIH3T3 cells.

The cells are expanded in T175 flasks and expanded further into multiple3 L spinner flasks until approximately 5×10⁸ cells are obtained.Subsequently, the cell lines are transferred to CELL-PHARM System 2000hollow fiber cell culture systems (Unisyn Technologies, Hopkinton,Mass.) for the production and continuous recovery ofbutyrylcholinesterase. The hollow fiber system permits high celldensities to be obtained (10⁸/ml) from which 60-120 ml of concentratedbutyrylcholinesterase is harvested each day. It is anticipated that itrequires one month to produce sufficient quantities ofbutyrylcholinesterase for further evaluation.

The concentrated recombinant butyrylcholinesterase harvested from thehollow fiber systems are purified, essentially as described previously(Masson et al., supra, 1997). The serum-free medium is centrifuged toremove particulates, its ionic strength is reduced by dilution with twovolumes of water, and subsequently, the sample is loaded on aprocainamide Sepharose affinity column. Butyrylcholinesterase is elutedwith procainamide, purified further by ion exchange chromatography andconcentrated. A recombinant butyrylcholinesterase mutant expressed inCHO cells has previously been enriched to 99% purity with over 50%yields using this purification approach (Lockridge et al., Biochemistry36:786-795 (1997)). The enzyme is filter-sterilized through a 0.22-μmmembrane and stored at 4° C. Under these conditions,butyrylcholinesterase retains over 90% of its original activity after 18months (Lynch et al., Toxicology and Applied Pharmacol. 55:83-91(1999)).

EXAMPLE VI Evaluation of Wild-Type Butyrylcholinesterase andButyrylcholinesterase Variants

This example describes the evaluation of wild-type butyrylcholinesteraseand butyrylcholinesterase variants in rat cocaine toxicity andreinforcement models.

Butyrylcholinesterase variants that display increased cocaine hydrolysisactivity in vitro display greater potency for the treatment of cocainetoxicity and addiction in vivo. To characterize thebutyrylcholinesterase variants in vivo, an acute overdose model is usedto measure the potency of butyrylcholinesterase variants for toxicity,while models of reinforcement and discrimination are used to predict thepotency of butyrylcholinesterase variants for the treatment ofaddiction. Although the pharmacokinetics of human butyrylcholinesterasevariants are not expected to be optimal in models, the rat cocainemodels are well characterized and require significantly smallerquantities of purified butyrylcholinesterase than do primate models. Itis anticipated that both wild-type butyrylcholinesterase and thebutyrylcholinesterase variants with increased cocaine hydrolysisactivity display dose-dependent responses. Furthermore, thebutyrylcholinesterase variant optimized for cocaine hydrolysis activityare efficacious at substantially smaller doses than the wild-typebutyrylcholinesterase.

Modification of the Toxicity of Cocaine

The effect of butyrylcholinesterase variants on cocaine toxicity isevaluated as previously described in rat model of overdose by Mets etal., Proc. Nat. Acad. Sci. USA 95:10176-10181 (1998). This model usesco-infusion of catecholamines because variable endogenous catecholaminelevels have been shown to affect cocaine toxicity (Mets et al., LifeSci. 59:2021-2031 (1996)). Infusion of cocaine at 1 mg/kg/min producesLD₅₀=10 mg/kg and LD₉₀=16 mg/kg when the levels of catecholamines arestandardized.

Six groups of six rats each are used in this study. The rats areSprague-Dawley males, weighing 250-275 g upon receipt in the vivarium,which is maintained on a 12 hour light-dark cycle. The rats have foodand water available ad libitum at all times. Prior to treatment the ratsare fitted with femoral arterial and venous catheters and permitted torecover. Subsequently, the rats are treated with varying amounts of thebutyrylcholinesterase variants (0.35, 1.76, or 11.8 mg/kg) or equivalentvolumes of saline 15 minutes prior to the co-infusion of catecholaminesand cocaine (1 mg/kg/min). The infusion is for 16 minutes to deliver theLD₉₀ of cocaine, unless the animals expire sooner. Based on the relativecatalytic efficiencies of wild-type butyrylcholinesterase and thepreviously described catalytic antibody (Mets et al., supra, 1998), itis anticipated that increasing doses of butyrylcholinesterase conferincreased survival rate to the rats relative to the saline controls andthat the highest butyrylcholinesterase dose (11.8 mg/kg) protects allthe animals. A butyrylcholinesterase variant that hydrolyzes cocaine10-fold more efficiently in vitro is be expected to confer protection toall of the animals at a lower dose (1 mg/kg, for example).

Modification of the Abuse of Cocaine

The discriminative and reinforcing pharmacological effects of cocaineare believed to most closely reflect the actions of cocaine that embodyabuse of the drug. Therefore, the butyrylcholinesterase variants areevaluated in both cocaine reinforcement and cocaine discriminationmodels in rats.

The rat model of the reinforcing effects of cocaine has been usedextensively to evaluate other potential therapies for cocaine (Koob etal., Neurosci. Lett. 79: 315-320(1987); Hubner and Moreton,Psychopharmacology 105: 151-156 (1991); Caine and Koob, J. Pharmacol.Exp. Ther. 270:209-218 (1994); Richardson et al., Brain Res. 619: 15-21(1993)).

Male Sprague-Dawley rats are maintained as described above. Six operantchambers (Med Associates, St. Albans, Vt.), equipped with a house light,retractable lever, dipper mechanism, red, yellow, and green stimuluslights, and a pneumatic syringe-drive pump apparatus (IITC LifeSciences, Inc., Woodland Hills, Calif.) for drug delivery are interfacedwith an IBM-compatible computer through input and output cards (MedAssociates, Inc., St. Albans, Vt.). The chambers are housed within anair conditioned, sound attenuating cubicle (Med Associates). Customself-administration programs, controlling scheduled contingencies andstimulus arrays within the operant chambers, are written using theMed-PC programming language for DOS.

The reinforcing effects of cocaine are assessed in a model thatquantitates the number of injections taken by rats under conditions inwhich intravenous administration is contingent upon a response made bythe animal (Mets et al., supra, 1998). The rats are trained in theoperant conditioning chambers to press a lever in order to gain accessto 0.5 ml of a sweetened milk solution. After the rats have acquired thelever-press response on a fixed-ratio 1 (FR1) schedule of reinforcement,the response requirements are successively increased to an FR5 schedule.When the rats display stable rates of milk-maintained responding overthree consecutive days on this schedule (less than 10% variability inreinforcer deliveries over the one-hour session) a catheter issurgically introduced in the left internal jugular vein and the rats aregiven a minimum of two days to recover from surgery.

On the first operant training session following surgery, rats areallowed to respond on the lever, in a one-hour session, for thesimultaneous 5-second delivery of both milk and an intravenous bolus ofcocaine (0.125 mg/kg/injection). The milk is then removed from thechamber and for the next three days, the rats are given access to one ofthree doses of cocaine (0.125, 0.25, or 0.5 mg/kg/injection) for onehour each, in self-administration sessions six hours in duration. Thus,the rats are allowed access to each dose twice per session and the dosesare presented in repeated ascending order (i.e., 0.125, 0.25, 0.5,0.125, 0.25, 0.5 mg/kg/injection). Within each one-hour longdose-component, the original FR5 schedule with a 10-second timeout isretained. In addition, 10-minute timeout periods are instituted aftereach dose component in an attempt to minimize carryover effects acrossthe individual one-hour sessions.

When the rats display consistent cocaine self-administration (over 160injections per six-hour session with less than 15% variability) overthree consecutive days, they are placed on a schedule in which smallerdoses, as well as saline, are available during single daily sessions.Each session is divided into two components, with saline and three dosesof cocaine available in each component. The first component of eachsession provides access to a series of low doses (0-0.0625mg/kg/injection) while the second component provides access to a widerrange of doses (0-0.5 mg/kg/injection).

After the rates of cocaine self-administration are stabilized the ratsare divided between six groups and each group (n=6 rats) is given 0.35,1.76, or 11.8 mg/kg of either wild-type butyrylcholinesterase, theoptimized butyrylcholinesterase variant or an equivalent volume ofsaline 30 minutes prior to the beginning of the dailyself-administration sessions. The effects of the pretreatment aremonitored for several days until the cocaine self-administrationbehavior of the rat returns to baseline.

Using a fixed ratio (FR) schedule, the number of injections is limitedonly by the duration of the session and consequently, the number ofinjections is used as the dependent variable to compare the potency ofoptimized butyrylcholinesterase with wild-type butyrylcholinesterase.Following administration of varying concentrations of wild-typebutyrylcholinesterase or the optimized butyrylcholinesterase variant,the dose response curves are analyzed using a mixed factor MANOVA. Thebutyrylcholinesterase concentration (0.35, 1.76, or 11.8 mg/kg) isloaded as the between-subjects factor and the cocaine dose (0, 0.015,0.03, 0.06, 0.125, 0.25, 0.5 mg/kg/injection) is loaded as thewithin-subjects factor. All individual comparisons acrossbutyrylcholinesterase treatment groups at individual cocaine doses usethe Tukey HSD post-hoc procedure (see Gravetter, F. J. and Wallnau, L.B., Statistics for the Behavioural Sciences (5th ed., 2000, WadsworthPubl., Belmont, Calif.)) and the criterion for statistical significanceis set at p<0.05. At higher butyrylcholinesterase doses (11.8 mg/kg),the number of injections taken by the rats is expected to be lower thanthe untreated (saline) control group. Furthermore, rats treated with thebutyrylcholinesterase variant displaying enhanced cocaine hydrolysis areexpected to reduce their number of injections at a smaller dose (0.35mg/kg) than the animals treated with the wild-typebutyrylcholinesterase.

Drug discrimination is relevant to the subjective effect of cocaine inclinical situations and antagonism of cocaine discrimination followingpretreatment is considered clear evidence of therapeutic potential(Holtzman, Modern Methods in Pharmacology, Testing and Evaluation ofDrug Abuse, Wiley-Liss Inc., New York, (1990); Spealman, NIDA Res. Mon.119: 175-179 (1992)). The most frequently used procedure to establishand evaluate the discriminative stimulus effect of drugs is to trainanimals in a controlled operant procedure to use the injected drug as astimulus to control distribution of responding on two levers.Dose-effect curves consisting of distribution of the responses on the“drug-associated” lever as a function of drug dose are easily generated.These cocaine dose-effect curves can be altered by the administration ofa competitive antagonist. The amount of the shift of the curve and timerequired for the original sensitivity of the animal to cocaine to returnare useful data for evaluating the potential therapeutic use ofwild-type butyrylcholinesterase and the optimized variant. Thediscriminative stimulus effects of cocaine in rat models have been usedto evaluate the therapeutic potential of dopamine reuptake inhibitors,as well as agonists and antagonists to the dopamine receptors (Witkin etal., J. Pharmacol. Exp. Ther. 257: 706-713 (1989); Kantak et al., J.Pharmacol. Exp. Ther. 274: 657-665 (1995); Barret and Appel,Psychopharmacology 99: 13-16 (1989); Callahan et al., Psychopharmacology103: 50-55 (1991)).

A multiple trial procedure for training and testing cocaine as adiscriminative stimulus is used to evaluate the potency ofbutyrylcholinesterase in rats as previously described in Bertalmio etal. J. Pharmacol. Methods 7: 289-299 (1982) and Schecter, Eur. J.Pharmacol. 326: 113-118 (1997). A dose-response curve for cocaine isobtained in a single session in the presence of butyrylcholinesterase orthe optimized butyrylcholinesterase variant. Subsequently, the recoveryof the rat's original sensitivity to cocaine is tracked on atwice-weekly basis to assess the duration of action of thebutyrylcholinesterase.

The rats are deprived to 80% of their free-feeding weight at thebeginning of the experiment in order to train them in thefood-reinforced operant procedure. Each rat is placed in an operantconditioning chamber equipped with two light stimuli and two retractablelevers, one on either side of a milk delivery system and trained topress on one of the levers to receive access to 0.5 ml of sweetenedcondensed milk. Once the rats have learned to respond on this lever, amultiple-trials procedure is initiated. Each session consists of 6trials with each trial lasting 15 minutes. The first 10 minutes of eachtrial are a blackout period, during which no lights are on andresponding has no consequence. This 10-minute period allows for drugabsorption in the subsequent testing phases of the study. The last 5minutes of each trial are a milk-reinforced period (FR5). Once the ratsrespond consistently and rapidly during the 5-minute response period(signaling period), cocaine is introduced into the procedure.

Initially, 10 mg/kg cocaine is given 10 minutes prior to the beginningof three of six weekly sessions. During these sessions, the“non-cocaine” lever (saline) previously extended is retracted and theother, “cocaine-associated,” lever is extended on the other side of themilk delivery cup. Responses (initially only a single response;eventually five responses) on this second lever result in milkpresentation if cocaine was administered prior to the session. The ratsare being trained to respond on the second lever if they detect theinteroceptive effects of the administered cocaine. Because cocaine'sinteroceptive effects are not believed to extend beyond 30 minutes, thesessions following cocaine administration lasts for only two trials (15minutes each). At this juncture the rats do not receive a cocaineinjection on three days of the week and on those days they arereinforced with milk (FR5) for responding on the available non-cocainelever during the signaling periods of six trials. On the remaining threedays of the week, the rats are given 10 mg/kg cocaine before thebeginning of the session and are reinforced for responding on theavailable cocaine lever during the signaling periods on each of twotrials.

Subsequently, each daily session is initiated with one to four trialswithout cocaine administration, followed by the administration of 10mg/kg cocaine. Thus, each session ends with two trials in whichresponding on the cocaine-appropriate lever is required for fooddelivery. Although only the “correct” levers are extended during thisphase, the critical step of making both levers available during theentire session is taken as soon as the animals learn to switch from thenon-cocaine to the cocaine lever within daily sessions. Subsequently,each session begins with a 10-minute blackout period followed bypresentation of both levers for five minutes. During the first 1 to 4trials of a daily session, no cocaine is given, and 5 consecutiveresponses on the non-cocaine lever result in food during this 5-minuteperiod. If the rat switches from one lever to the other or responds onthe incorrect lever, he does not get reinforced and both levers areretracted for 10 seconds, at which time the levers are presented againand the trial restarted. At the start of the second, third, or fourthtrial, 10 mg/kg cocaine are given and the rat is returned to the testbox. When the light is illuminated and the levers presented on the nexttwo trials, five consecutive responses on the cocaine lever are requiredfor milk presentation to demonstrate that the rats are learning toswitch their responding from the non-cocaine lever to the cocaine leverusing the interoceptive effects of cocaine as a cue to tell them whichlever is correct on a given trial.

A cocaine dose-effect curve is obtained as soon as the rats meetcriterion of 80% correct lever selection on three consecutive sessions.On the first trial of a test session, saline is given. On subsequenttrials, 0.1, 0.3, 1.0, 3.2, and 10 mg/kg cocaine is administered, eachat the start of the 10 minute blackout that begins each trial. Duringthese test trials, five consecutive responses on either lever result inmilk presentation, but switching from one lever to the other prior tocompletion of an FR results in lever retraction for 10 seconds. It isanticipated that animals begin this session with responses on thenon-cocaine lever and gradually increase the percent of responses madeon the cocaine lever until all responses are made on that lever. Thus, adose-response curve of lever selection versus dose of cocaineadministered is established during each test session.

Once cocaine has been established as a discriminative stimulus, the ratsare placed in separate groups (n=6 per group) that receive 0.35, 1.76,or 11.8 mg/kg of either wild-type butyrylcholinesterase or the optimizedvariant. The discriminative stimulus effects of cocaine is determined 30minutes following enzyme administration and daily afterwards untilsensitivity to cocaine is re-established. On the initial test sessionfollowing administration of butyrylcholinesterase, larger doses ofcocaine are given if there is no selection of the cocaine leverfollowing any of the smaller test doses. Doses as large as 100 mg/kgcocaine are given if the animals fail to select the cocaine-appropriatelever following administration of 10 or 32 mg/kg cocaine. Becausedose-response curves to cocaine can be obtained in a single session,this protocol provides information on the relative ability of the twotypes of butyrylcholinesterase to decrease the potency of cocaine as adiscriminative stimulus, which is a relevant aspect of its abuseliability. The butyrylcholinesterase variant displaying enhanced cocainehydrolysis activity in vitro is more potent.

EXAMPLE VII In Vivo Confirmation of the Therapeutic Effect ofButyrylcholinesterase Variant on Cocaine-Toxicity

This example describes the confirmation, in vivo, of the therapeuticeffect of butyrylcholinesterase variants provided by the invention oncocaine toxicity.

Briefly, unilateral/bilateral jugular cannulations were performed underisoflurane anesthesia (max. 20 min) on 350 g male Sprague Dawley®SD®rats (Harlan Sprague Dawley, Inc.) The animals were allowed to recoverfor 24 hours before studies commenced. For in vivo confirmation of thetherapeutic effect on cocaine toxicity of butyrylcholinesterase variantdesignated A328W/Y332M/S287G/F227A (SEQ ID NO: 52), wild-type BChE(10-50 mg/kg) or the A328W/Y332M/S287G/F227A variant (0.1-0.5 mg/kg) wasinjected intravenously, then flushed with 200 ml saline. One minutelater, the cannula was connected to an infusion pump (Harvard Apparatus)and cocaine (HCl salt) was infused at 6 mls/hr (2 mg/kg/min) for 15minutes corresponding to a total dose equal to 30 mg/kg. Thetime-to-onset for slight convulsions, strong convulsions and death wererecorded. In pilot studies, the lethal dose of cocaine was determined tobe approximately 25 mg/kg. In a second series of studies, 0.5 mg/kg ofthe A328W/Y332M/S287G/F227A variant was administered at various timepoints after initiation of the 30 mg/kg cocaine infusion and grossobservations recorded. As demonstrated in FIG. 8, which shows the effectof pre-treatment with the A328W/Y332M/S287G/F227A variant (solidcircles) or wild-type BChE (open circles) on cocaine-induced toxicity,the A328W/Y332M/S287G/F227A variant exhibited statistically significantprotection against cocaine (Chi-squared test; p<0.001).

FIG. 9 further demonstrates the therapeutic effect on cocaine-inducedtoxicity of the variant, by showing that the A328W/Y332M/S287G/F227Avariant provided full protection when administered at 8 minutes into thecocaine infusion (i.e. after the first set of slight convulsions) anddecreased in ability to protect when administered at later time points.

For pharmacokinetic studies cannulated rats were injected with wild-typeBChE and the A328W/Y332M/S287G/F227A variant at 1 mg/kg and plasma wascollected at the indicated timepoints. Plasma was analyzed for BChEactivity using a standard assay for BChE utilizing butyrylthiocholine asthe substrate. In a separate set of studies, a 10 mg/kg i.v. bolus ofcocaine was administered followed immediately by theA328W/Y332M/S287G/F227A variant (0.01, 0.02 or 0.5 mg/kg) and plasmacollected at the indicated time points. Circulating cocaine levels inthese samples were determined by ELISA (Immunalysis, Pomona, Calif.).FIG. 10 shows the plasma levels of wt BChE and theA328W/Y332M/S287G/F227A variant following an intravenous bolus of 1mg/kg. Wild-type BChE pool I or pool II (open squares and open circles,respectively) and the A328W/Y332M/S287G/F227A variant pool I or pool II(solid squares and solid circles, respectively). BChE activity wasdetermined by enzymatic assay utilizing butyrylthiocholine as thesubstrate.

FIG. 11 shows plasma levels of an intravenous bolus of Cocaine aftertreatment with the A328W/Y332M/S287G/F227A variant. Cocaine wasadministered at 10 mg/kg (open circles) and the A328W/Y332M/S287G/F227Avariant administered immediately at 0.01 mg/kg and 0.05 mg/kg (solidcircles and solid squares, respectively). Plasma samples were collectedat the indicated time points and analyzed for cocaine levels by ELISA.

FIG. 12 shows the effect of pre-treatment with theA328W/Y332M/S287G/F227A variant on time-to-onset for slight convulsions.The variant was administered at the indicated doses, 1 minute prior toinfusion of 30 mg/kg cocaine (2 mg/kg/min for 15 minutes). The data inFIG. 12 is presented as mean±sem.*p<0.001 vs. control, 0.1 mg/kg or 0.2mg/kg variant-treated animals; ANOVA followed by Bonferroni post-test.

This example demonstrates the therapeutic effect of theA328W/Y332M/S287G/F227A variant (SEQ ID NO: 52) on cocaine-toxicity forpre-treatment as well as for treatment subsequent to cocaine exposure.

Throughout this application various publications have been referenced.The disclosures of these publications in their entireties are herebyincorporated by reference in this application in order to more fullydescribe the state of the art to which this invention pertains.

Although the invention has been described with reference to thedisclosed embodiments, those skilled in the art will readily appreciatethat the specific experiments detailed are only illustrative of theinvention. It should be understood that various modifications can bemade without departing from the spirit of the invention. Accordingly,the invention is limited only by the following claims.

1. A butyrylcholinesterase variant polypeptide comprising an amino acid sequence set forth as SEQ ID NO:
 52. 2. A nucleic acid encoding a butyrylcholinesterase variant polypeptide comprising an amino acid sequence set forth as SEQ ID NO:
 52. 3. A nucleic acid encoding a butyrylcholinesterase variant polypeptide comprising a nucleic acid sequence set forth as SEQ ID NO:
 51. 4. A method of treating a cocaine-induced condition comprising administering to an individual an effective amount of the butyrylcholinesterase variant polypeptide of claim
 1. 5. The method of claim 4, wherein said cocaine-based substance is cocaine.
 6. The method of claim 5, wherein said individual is symptomatic of a cocaine-overdose.
 7. The method of claim 5, wherein said individual is symptomatic of cocaine addiction.
 8. A method of hydrolyzing a cocaine-based butyrylcholinesterase substrate comprising contacting said butyrylcholinesterase substrate with the butyrylcholinesterase variant polypeptide of claim 1, under conditions that allow hydrolysis of cocaine into metabolites, wherein said butyrylcholinesterase variant polypeptide exhibits a one-hundred-fold or more increase in cocaine hydrolysis activity compared to butyrylcholinesterase.
 9. A method of treating a cocaine-induced condition comprising administering to an individual an effective amount of a butyrylcholinesterase variant polypeptide of claim 1, wherein said butyrylcholinesterase variant polypeptide exhibits a one-hundred-fold or more increase in cocaine hydrolysis activity compared to butyryleholinesterase.
 10. The method of claim 9, wherein said cocaine-based substance is cocaine.
 11. The method of claim 10, wherein said individual is symptomatic of a cocaine-overdose.
 12. The method of claim 10, wherein said individual is symptomatic of cocaine addiction. 